Synthesis of locked nucleic acid derivatives

ABSTRACT

The invention relates to a novel strategy for the synthesis of Locked Nucleic Acid derivatives, such as α- L -oxy-LNA, amino-LNA, α- L -amino-LNA, thio-LNA, α- L -thio-LNA, seleno-LNA and methylene LNA, which provides scalable high yielding reactions utilising intermediates that also can produce other LNA analogues such as oxy-LNA. Also, the compounds of the formula X are important intermediates that may be reacted with varieties of nucleophiles leading to a wide variety of LNA analogues.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority to U.S. application Ser. No.12/534,711, filed Aug. 3, 2009, as a divisional application which claimsthe priority to U.S. application Ser. No. 10/435,607, filed May 8, 2003,as a divisional application. U.S. application Ser. No. 10/435,607 claimsthe benefit of U.S. Provisional Application Ser. No. 60/378,689, filedMay 8, 2002 and 60/404,242 filed Aug. 16, 2002, all of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a novel strategy for the synthesis ofLocked Nucleic Acid derivatives, such as amino-LNA, thio-LNA, seleno-LNAand methylene-LNA, which provides scalable high yielding reactionsutilising intermediates that also can produce other LNA analogues suchas oxy-LNA. The invention further relates to a novel strategy for thesynthesis of α-L-LNA analogues and precursors.

BACKGROUND OF THE INVENTION

Professor Imanishi (WO 98/39352) and Professor Wengel (WO 99/14226)independently invented Locked Nucleic Acid (LNA) in 1997 and the firstLNA monomer was based on the 2′-O—CH₂-4′ bicyclic structure (oxy-LNA).This LNA analogue has since then showed promising results as antisensedrug candidates. Other LNA analogues has also been synthesizedexhibiting similar high affinity/specificity for example 2′—NH—CH₂-4′,2′—N(CH₃)— CH₂-4′ (amino-LNA) (Singh, S. K.; Kumar, R.; Wengel, J. J.Org. Chem. 1998, 63, 10035-10039; Singh, S. K.; Kumar, R.; Wengel, J. J.Org. Chem. 1998, 63, 6078-6079), and 2′-S—CH₂-4′ (thio-LNA) (Singh, S.K.; Kumar, R.; Wengel, J. J. Org. Chem. 1998, 63, 6078-6079, Kumar, R.;Singh, S. K et al. Biorg. Med. Chem. Lett. 1998, 8, 2219-2222). Largequantities of amino-LNA are crucial for its use in antisense. Scaling-upthe previously described method of synthesis of amino-LNA has appearedto be difficult and encountered several major problems.

The first difficult reaction in the scale up work proved to be theregioselective benzylation of3-O-benzyl-1,2-O-isopropylidene-4-C-hydroxynnethyl-α-D-erythro-pentofuranose(Koshkin, A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.; Kumar, R.;Meldgaard, M.; Olsen, C. E.; Wengel, J. Tetrahedron 1998, 54, 3607-3630)(see FIG. 1, compound 1).

Working in the 100 g range the reaction yielded a product-mixture ofcompound 2, the 1′-benzylated and the di-benzylated material even underoptimised conditions. The maximum yield of the desired compound 2 was59% dropping to an average of 45-50% compared to 71% on smaller scale.Furthermore, compound 2 could only be isolated through tediouschromatography of closely eluting products.

The second key step in the original strategy causing problems duringscale-up synthesis was the double nucleophilic substitution of thedi-O-tosyl nucleoside 5 using benzylamine giving nucleoside 6 (Singh, S.K.; Kumar, R.; Wengel, J. J. Org. Chem. 1998, 63, 10035-10039). Thereaction on larger scale (22 g) apparently afforded a second productidentified as the oxy-LNA derivative. The desired N-benzylated-amino-LNAproduct 6 was obtained in only 15% together with 13% of the oxy-LNAby-product. For comparison, the reaction gives 52% of nucleoside 6 on a8 g scale with no side reaction reported (Singh, S. K.; Kumar, R.;Wengel, J. J. Org. Chem. 1998, 63, 10035-10039).

Yet another problem encountered appeared to be the debenzylation ofnucleoside 6 using ammonium formate and 10% Pd/C in methanol. Itappeared to be only partial debenzylation as verified by massspectroscopy, and the product 7 proved to be difficult to isolate fromthe reaction mixture.

The first synthesis of an oxy-LNA nucleoside was performed by a linearapproach using uridine as starting material (Obika, S.; Nanbu, D.; Hari,Y.; Morio, J. A. K.; In, Y.; Ishida, T.; Imanishi, T. Tet. Lett. 1997,38, 8735-8738) but by virtue of being a convergent synthesis the routedeveloped by Wengel and coworker (Koshkin, A.; Singh, S. K.; Nielsen,P.; Rajwanshi, V. K.; Kumar, R.; Meldgaard, M.; Olsen, C. E.; Wengel, J.Tetrahedron 1998, 54, 3607-3630; Koshkin, A. A. et al., J. Org. Chem.2001, 66, 8504-8512) became the method of choice for the synthesis ofLNA nucleosides.

Amino- and thio-LNA was originally synthesised quite differently, butaccording to the present invention there are common intermediates thatcan be used for amino-LNA, thio-LNA, seleno-LNA, α-L-LNA as well asmethylene-LNA at late stages in the overall synthesis.

SUMMARY OF THE INVENTION

The present invention provides a novel strategy for the synthesis of LNAderivatives, such as α-L-oxy-LNA, amino-LNA, α-L-amino-LNA, thio-LNA,α-L-thio-LNA, seleno-LNA and methylene-LNA.

The compounds of the formula I are important intermediates that may bereacted with varieties of nucleophiles leading to a wide variety of LNAanalogues, e.g. amino-LNA, thio-LNA, seleno-LNA and methylene-LNA.

One aspect of the invention relates to a method for synthesis of LNAanalogues of the formula IV starting from compounds of formula I, cf.claim 1.

Another aspect of the present invention relates to the novel compounds(intermediates) of the formula I as defined in claim 33.

Still another aspect of the present invention relates to a method forsynthesis of the compounds (intermediates) of the formula I, cf. claim19.

A further object of the invention is to provide a method for thesynthesis of α-L-LNA analogues of the formula VIII, from an intermediateof the general formula IX, cf. claim 45.

The main advantages of the present invention comprises the following:

-   -   tedious separation of regioisomers is eliminated,    -   the low-yielding step of double nucleophilic substitution of        di-O-tosyl nucleoside using benzylamine is avoided,    -   the method enables the utilisation of a starting intermediates        which is common to the known oxy-LNA synthesis,    -   the method comprises a novel intermediate that when reacted with        appropriate nucleophilic can produce a variety of LNA analogues,        i.e. amino-LNA, thio-LNA, seleno-LNA, methylene-LNA, and        α-L-LNA,    -   the method comprises an alternative method for N-methylation,        and hereby avoids methylation at the nucleobase,    -   employs cheap and commercial available reagents,    -   comprises scalable reactions giving access to large quantities        of LNA analogue phosphoramidites.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a known method for the preparation of amino-LNAaccording to Singh, S. K.; Kumar, R.; Wengel, J. J. Org. Chem. 1998, 63,10035-10039.

FIG. 2 illustrates the generalised method for the preparation of the LNAanalogues.

FIG. 3 illustrates inversion at C2′ for a compound not having apyrimidine base.

FIG. 4 illustrates a further alternative for inversion at C2′.

FIG. 5 illustrates the synthesis of a preferred compound of the formulaVII. The known compound1-(2-O-acetyl-3-O-benzyl-4-C-methanesulfonyloxymethyl-5-O-methane-sulfonyl-β-D-erythro-pentofuranosyl)thymine (23) is converted by a mild deacetylation for the liberation ofthe 2′-hydroxy group to the compound (24) without the subsequentring-closure that affords the oxy-LNA skeleton. The 2′-hydroxy group isthen mesylated to1-(3-O-benzyl-4-C-methanesulfonyloxymethyl-2,5-O-dimethanesulfonyl-β-D-erVthro-pentofuranosyl)thymine (25). Legend: i) 50% methanolic ammonia; ii) MsCl, pyridine.

FIG. 6 illustrates a preferred example for the preparation of twoamino-LNA phosphoramidite that are useful in the preparation ofoligonucleotides. Legend: i) half sat. NH₃ in MeOH; ii) MsCl, anh.pyridine, anh. CH₂Cl₂; iii) DBU, DMF; iv) acetone, 0.1M H₂SO₄; v) Tf₂O,DMAP, anh. pyridine, anh. CH₂Cl₂; vi) NaN₃, anh. DMF; vii) PMe₃, NaOH(aq), THF; viii) CH₂O, HCO₂H; ix) a) NaOBz, DMF, b) NaOMe; x) 20%Pd(OH)₂/C, H₂, AcOH; xi) DMT-Cl, anh. pyridine; xii)NC(CH₂)₂OP(N(iPr)₂)₂, DCl, CH₃CN, CH₂Cl₂. xiii) Ac₂O, pyridine; xiv)Et₃N, 1,2,4-triazole, POCl₃, MeCN; xv) 1:1 MeCN, sat. aq NH₃; xvi) BzCl,pyridine; xvii) LiOH (aq), THF.

FIG. 7 illustrates further acylation and alkylation of the 2′-aminogroup of amino-LNA

FIG. 8 illustrates a preferred example for the preparation of a thio-LNAphosphoramidite that is useful in the preparation of oligonucleotides.Legend: i) Pd/C, H₂, Acetone, MeOH; ii) BzCl, Pyridine, DMF; iii) 0.25 Maq. H₂SO₄, DMF, 80° C.; iv) Tf₂O, DMAP, CH₂Cl₂, 0° C.; v) Na₂S, DMF; vi)NaOBz, DMF, 100° C.; vii) NH₃, MeOH; viii) DMT-Cl, Pyridine; ix)P(OCH₂CH₂CN)(N(i-Pr)₂)₂, 4,5-Dicyanoimidazole, CH₂Cl₂.

FIG. 9 illustrates the synthesis of an amino-LNA analogue 33 and athio-LNA analogue 60 from the key intermediate 31 (a preferred exampleof a compound of Formula I). Legend: i) potassium thioacetate, DMF, ii)sodium azide in DMF, iii) LiOH in THF, iv) NaOH (aq.), Me₃P, THF.

FIG. 10 illustrates the synthesis of the α-L-oxy-LNA A (63),α-L-amino-LNA A (65), as well as the synthesis of an epoxide (66) fromthe key intermediate 62, which is opened up with different nucleophilesto form either an azide (67) or a thio-LNA (68). Legend: i) Tf₂O,pyridine, DCM, ii) LiOH, aq, THF, iii) NaN₃, DMF, iv) NaOH, PMe₃, THF,v) MsOH, DCM, vi) Na₂S, DMF.

FIG. 11 illustrates a preferred example for the preparation of anα-L-thio-LNA phosphoramidite that is useful in the preparation ofoligonucleotides. Legend: i) Na₂S, DMF, ii) NaOBz, DMSO, 100° C., iii)MsOH, DCM, iv) LiOH, aq, THF, v) DMT-CI, DMAP, Pyridine vi)P(OCH₂CH₂CN)—(N(i-Pr)₂)₂, 4,5-Dicyanoimidazole, CH₂Cl₂.

FIG. 12 illustrates a preferred example for the preparation of anα-L-LNA-G phosphoramidite that is useful in the preparation ofoligonucleotides. Legend: i) BSA, TMSOTf, ClCH₂CH₂Cl, ii) half sat.methanolic NH₃, iii) Tf₂O, DMAP, pyridine, CH₂Cl₂, iv) HOCH₂CH₂CN, NaH,THF; v) NaOBz, DMSO; vi) NH₄HCO₂, Pd(OH)₂—C, MeOH; vii)(CH₃O)₂CHN(CH₃)₂, DMF; viii) DMT-Cl, pyridine, ix) NC(CH₂)₂OP(N(iPr)₂)₂,4,5-dicyanoimidazole, MeCN, CH₂Cl₂, x) LION, aq, THF.

FIG. 13 illustrates particularly interesting compounds according to theinvention.

DETAILED DESCRIPTION OF THE INVENTION

Synthesis of LNA Analogues

A main aspect of the present invention relates to a method for thesynthesis an LNA analogue of the general formula IV

wherein

-   X is selected from —CH₂—, —NR^(H)—, —O—, and —S—;-   Z is selected from —CH₂—, —NR^(H)—, —S—, and —Se—;-   B is a nucleobase;-   R³ is selected from —R^(H), —N₃, —NR^(H)R^(H)*, —NR^(H)C(O)R^(H)*,    —C(O)NR^(H)R^(H)*, —OR^(H), —OC(O)R^(H), —C(O)OR^(H), —SR^(H),    —SC(O)R^(N), and tri(C₁₋₆-alkyliaryl)silyloxy;

each R^(H) and R^(H)* independently being selected from hydrogen,optionally substituted C₁₋₆-alkyl, optionally substituted aryl, andoptionally substituted aryl-C₁₋₆-alkyl;

-   A⁴ and A⁵ independently are selected from C₁₋₆-alkylene; and-   R⁵ is selected from iodo, bromo, chloro, C₁₋₆-alkylsulfonyloxy    optionally substituted with one or more substituents selected from    halogen and phenyl optionally substituted with one or more    substituents selected from nitro, halogen and C₁₋₆-alkyl, and    arylsulfonyloxy optionally substituted with one or more substituents    selected from nitro, halogen, C₁₋₆-alkyl, and C₁₋₆-alkyl substituted    with one or more halogen;

said method comprising the following steps:

treating an intermediate of the general formula I:

wherein

-   X, B, R³, A⁴, and A⁵ are as defined above;-   R² is selected from iodo, C₁₋₆-alkylsulfonyloxy optionally    substituted with one or more substituents selected from halogen and    phenyl optionally substituted with one or more substituents selected    from nitro, halogen and C₁₋₆-alkyl, and arylsulfonyloxy optionally    substituted with one or more substituents selected from nitro,    halogen, C₁₋₆-alkyl, and C₁₋₆-alkyl substituted with one or more    halogen; R³ and R² may together form an epoxide and R⁴ and R⁵    independently are as defined for R⁵ above, or R⁴ and R⁵ together    constitutes a tetra(C₁₋₆-alkyl)disiloxanylidene group;

with a nucleophile selected from halogen, ⁻N₃, ⁻NR^(H)R^(H)*, ⁻SR^(H),⁻⁻S, ⁻SeR^(H), ⁻⁻Se ⁻NR^(H)C(O)R^(H)*, ⁻SC(O)R^(H), and organometallichydrocarbyl radicals, so as to substitute R², and

effecting ring-closure between the C2′ and C4′ positions so as to yieldthe LNA analogue of the formula IV.

It has been found that the intermediates of the formula I play animportant role in the synthesis of the LNA analogues. Hence, theparticular selection of substituents in the intermediates has proved tobe important for the efficient route to the LNA analogues. It should beunderstood that the substituents X, B, R³, A⁴, A⁵, and R⁵ most oftenwill be unaltered in the synthesis, i.e. these substituents will be“carried over” from Formula I to formula IV. Also the absoluteorientation of these substituents will also be preserved.

This being said, it may be necessary to protect the nucleobase as willbe appreciated by the person skilled in the art (see further below underthe definition of “nucleobase” and FIG. 3).

In an interesting embodiment, the substituents of the compound of theformula I are selected so that

R² is selected from C₁₋₅-alkylsulfonyloxy optionally substituted withone or more substituents selected from halogen and phenyl optionallysubstituted with one or more substituents selected from nitro, halogenand C₁₋₆-alkyl, and arylsulfonyloxy optionally substituted with one ormore substituents selected from nitro, halogen, C₁₋₆-alkyl, andC₁₋₆-alkyl substituted with one or more halogen;

-   R³ is optionally substituted aryl(C₁₋₆-alkyl)oxy; and-   R⁴ and R⁵ are independently selected from C₁₋₆-alkylsulfonyloxy    optionally substituted with one or more substituents selected from    halogen and phenyl optionally substituted with one or more    substituents selected from nitro, halogen and C₁₋₅-alkyl, and    arylsulfonyloxy optionally substituted with one or more substituents    selected from nitro, halogen, C₁₋₅-alkyl, and C₁₋₆-alkyl substituted    with one or more halogen.

Also, interesting is the embodiments where A⁴ and A⁵ are both methylene,as well as the embodiments where X is —O—.

Although the configuration of the intermediate (Formula I) is generallyopen, it is presently believed that one interesting configuration forthe intermediate is represented by the formula II

wherein B, R², R³, R⁴, and R⁵ are as defined above. This being said, themirror-image of formula II may be equally applicable. In one embodimentOR³ and R² may form an epoxide.

In a particularly interesting embodiment, the substituents of theintermediate (Formula I or Formula II) are chosen so that B is selectedfrom adenine, guanine, 2,6-diaminopurine, thymine, 2-thiothymine,cytosine, methyl cytosine, uracil, 5-fluorocytosine, xanthine,6-aminopurine, 2-aminopurine, 6-chloro-2-amino-purine, and6-chloropurine, R² is selected from C₁₋₅-alkylsulfonyloxy substitutedwith one or more halogen, R³ is benzyl, and R⁴ and R⁵ are independentlyselected from C₁₋₆-alkylsulfonyloxy optionally substituted with one ormore substituents selected from halogen and phenyl optionallysubstituted with one or more substituents selected from nitro, halogenand C₁₋₆-alkyl, and arylsulfonyloxy optionally substituted with one ormore substituents selected from nitro, halogen, C₁₋₆-alkyl, andC₁₋₆-alkyl substituted with one or more halogen.

The substituents R⁴ and R⁵ are preferably identical in that offersadvantages in the preparation of the intermediate (see further below).

Particular examples of the groups (independently) applicable as R⁴ andR⁵ are methanesulfonyloxy, trifluoromethanesulfonyloxy,ethanesulfonyloxy, 2,2,2-trifluoro-ethanesulfonyloxy,propanesulfonyloxy, iso-propanesulfonyloxy, butanesulfonyloxy,nona-fluorobutanesulfonyloxy, pentanesulfonyloxy,cyclopentanesulfonyloxy, hexanesulfonyloxy, cyclohexanesulfonyloxy,α-toluenesulfonyloxy, 2-chloro-α-toluenesulfonyloxy,ortho-toluenesulfonyloxy, meta-toluenesulfonyloxy,para-toluenesulfonyloxy, benzenesulfonyl-oxy,ortho-bromobenzenesulfonyloxy, meta-bromobenzenesulfonyloxy,para-bromo-benzenesulfonyloxy, ortho-nitrobenzenesulfonyloxy,meta-nitrobenzenesulfonyloxy, and para-nitrobenzenesulfonyloxy. Thecurrently most promising group is methanesulfonyloxy.

In one particularly interesting variant, the intermediate has theformula III

wherein B, R³, R⁴ and R⁵ are as defined above.

A further interesting variant (in combination with Formula I, Formula IIor Formula III) is where the substituents are chosen so that B isselected from adenine, guanine, 2,6-diaminopurine, thymine,2-thiothymine, cytosine, methyl cytosine, uracil, 5-fluorocytosine,xanthine, 6-aminopurine, 2-aminopurine, 6-chloro-2-amino-purine, and6-chloropurine, R³ is benzyl, and R⁴ and R⁵ are both methanesulfonyloxy.In particular, A⁴ and A⁵ are preferably both methylene.

The intermediate of Formula I is reacted with a nucleophile selectedfrom halogen, ⁻N₃, ⁻NR^(H)R^(H)*, ⁻SR^(H), ⁻⁻S, ⁻NR^(H)C(O)R^(H)*,—SC(O)R^(H), and organometallic hydrocarbyl radicals, so as tosubstitute R².

It is currently believed that the substitution of R² proceeds via aS_(N)2 mechanism with inversion of the relative orientation of thesubstituent in the C2′ position.

The “C2′ position” refers to the normal nomenclature for nucleosides,where the carbon carrying the nucleobase B is C1′, the carbon carryingR² (or R^(2*)) is C2′, and the carbon carrying R⁴A⁴ is C4′.

The organometallic hydrocarbyl radicals typically has the formula MR^(H)where M is a metal such as Mg (e.g. in the form 12″MgBr prepared fromthe halide and magnesium (Grignard)), Cu (R^(H) ₂CuLi e.g. prepared from2R^(H)Li+CuI), Li (e.g. BuLi)), etc.

The organometallic hydrocarbyl radicals are applicable for thepreparation of LNA analogues where Z is —CH₂— (methylene-LNA). Thesulphur nucleophiles are of course applicable where Z is —S—, and thenitrogen nucleophiles are applicable where Z is —NR^(H)—.

This being said, it is currently believed that particularly interestingnucleophile are those selected from ⁻N₃, ⁻NR^(H)R^(H)*, ⁻SR^(H), ⁻⁻S,⁻NR^(H)C(O)R^(H)*, and ⁻SC(O)R^(H).

The conditions for the reaction of the compound of Formula I with anucleophile is typically so that the temperature is 0-150° C., such as20-100° C., the reaction time is typically 5 min to 24 hours, such as2-8 hours, and the molar ratio of the nucleophile to the compound of theFormula I is typically in the range of 10:1 to 1:1, such as in the rangeof 5:1 to 1:1. The solvent used for the reaction is typically a polaraprotic solvent.

Examples of useful polar aprotic solvents for this reaction aretetrahydrofuran (THF), dimethylformamide (DMF), dimethylsulfoxide(DMSO), acetonitrile (AcCN), diethylether, etc.

After substitution of the group R² with the nucleophile, the (new) groupin the C2′ position (i.e. the nucleophile attached to the C2′ position)is subjected to such conditions that ring-closure between the C2′ andC4′ positions is effected so as to yield the LNA analogue of the formulaIV. The exact conditions for effecting ring closure will depend on thenucleophile used, or rather the (new) group in the C2′ position.

The conditions for the ring-closure reaction is typically so that thetemperature is 0-100° C., such as 20-50° C., and the reaction time istypically 5 min to 24 hours, such as 2-8 hours. The solvent used for thereaction is typically a polar solvents.

Examples of such polar solvents are DMF, THF, acetonitrile, DMSO,C₁₋₄-alcohols and aqueous mixtures thereof.

The reagent useful for facilitating the ring-closure is typically underbasic conditions using bases such as hydroxides, alkoxides, amines,deprotonated amines, etc.

In particular, in the embodiments where Z is —S—, Na₂S (of the type S⁻⁻)is a useful nucleophile that facilitates both substition and ringclosure(see preparation of 54). The temperature is typically 0-100° C., such as15-40° C., the reaction time is typically 5 min to 18 hours, such as 10min to 4 hours, and the molar ratio of the nucleophile to the compoundof the Formula I is typically in the range of 10:1 to 1:1, such as inthe range of 2:1 to 1:1. The polar aprotic solvent is typically DMF,THF, DMSO, acetonitrile, pyridine, N-methyl pyrrolidone (NMP),hexamethylphosphoramide (HMPA), etc.

In an other embodiment potassium thioacetate (of the ⁻SC(O)R^(H) type)is a useful nucleophile. In this instance, the ring-closure can beeffected under the influence of lithium hydroxide in a polar aproticsolvent (see preparation of 60). The temperature is typically 0-100° C.,such as 15-40° C., the reaction time is typically 5 min to 18 hours,such as 5 min to 2 hours, and the molar ratio of the nucleophile to thecompound of the Formula I is typically in the range of 10:1 to 1:1, suchas in the range of 3:1 to 1:1. The polar aprotic solvent is typicallyDMF, THF, DMSO, acetonitrile, pyridine, N-methylpyrrolidone (NMP),hexamethylphosphoramide (HMPA), etc.

In the embodiment where Z is —NH—, sodium azide is a useful nucleophile.In this instance, the ring-closure is effected under the influence ofsodium hydroxide and trimethyl-phosphane in a polar aprotic solvent. Thetemperature is typically 0-50° C., such as 15-30° C., the reaction timeis typically 1-24 hours, such as 2-8 hours, and the molar ratio of thenucleophile to the compound of the Formula I is typically in the rangeof 10:1 to 1:1, such as in the range of 5:1 to 1:1. The polar aproticsolvent is typically DMF, THF, DMSO, acetonitrile, pyridine,N-methylpyrrolidone (NMP), hexannethylphosphoramide (HMPA), etc.

When the resulting LNA analogues is one where Z is —NH—, the inventorshave found that it is possible to convert the LNA analogues into anotherLNA analogue where the nitrogen is alkylated by reaction with analkanal. Thus, the method may in this instance (Z═—NH—) furthercomprises the step of converting the LNA analogue wherein Z is —NH— toan LNA analogues where Z is —N(C₁₋₆-alkyl)- or N(aryl) by reacting asolution of the former LNA analogue with a reducing agent and aC₁₋₆-alkanal or an aromatic aldehyde or where Z is N(acyl) by reactingwith an acid chloride or an acid anhydride. Preferably where the aldehydis formaldehyde, benzaldehyde, pyrene-1-carbaldehyde, orphthalimidoacetaldehyde and the reducing agent is NaBCNH₃, or whereinthe acid chloride is benzoyl chloride or pyren-1-ylcarbonyl chloride(see FIG. 7). The method of the invention relates not only to thecompounds of Formula IV but equally to amino-LNA analogues in general.

Amino-LNA analogues are particularly interesting compounds of theinvention. For example, 9-mers oligonucleotides mixed sequencecontaining two or three of the novel modified 2′-amino-LNA monomers45-49 (see FIG. 7) hybridize efficiently and in general with very highthermal stabilities comparable with those obtained for the LNA orN-methyl 2′-amino-LNA references (ΔT_(m)/° C. in a thermal denaturationassay towards complementary RNA compound calculated per monomer:45=+9.1, 46=+7.3, 47=+6.5, 48=+3 and 49=+7). Also, a (almost) fullymodified N-benzoyl 2′-amino-LNA 9-mers oligonucleotides shows remarkablyefficient binding towards DNA and RNA complements (T_(m)/° C. 75 and 73,ΔT_(m)/° C. +6.3 and +6.1).

The triflate for Formula III is particularly useful as an intermediatefor a wide range of LNA analogues by reaction with appropriatenucleophiles. As an example, the triflate 31 (see FIG. 9) is used in thesynthesis of thio-LNA (2-oxo-5-thiobicyclo[2.2.1]heptane skeleton)accomplished by a substitution reaction with the nucleophile potassiumthioacetate in DMF producing compound 59. Ring-closure of the thio-LNAnucleoside was achieved by hydrolysis of the thioacetate with aq. LiOHin THF to produce 60 in quantitative yield. The structure of 60 wasconfirmed by NOE experiments showing an unusually high NOE effectbetween H6 of the nucleobase and H3′ (9.0%) as expected due to theextreme north conformation adopted by the nucleoside. Similarly,reaction of the triflate 31 with the nucleophile sodium azide in DMFproduced compound 32 which was subsequently ring-closed to the amino-LNAnucleoside 33 under the influence of aqueous sodium hydroxide andtrimethylphosphane in THF.

In one particularly interesting embodiment of intermediates of formulaI, R³ and R² together form an epoxide. Within the embodiment whereinformula I is such that R³ and R² together form an epoxide, A⁴ and A⁵independently are selected from C₁₋₆-alkylene; and R⁵ isC₁₋₆-alkylsulfonyloxy optionally substituted with one or moresubstituents selected from halogen and phenyl optionally substitutedwith one or more substituents selected from nitro, halogen andC₁₋₆-alkyl, and arylsulfonyloxy optionally substituted with one or moresubstituents selected from nitro, halogen, C₁₋₆-alkyl, and C₁₋₆-alkylsubstituted with one or more halogen; such as compound 66 in FIG. 10.

Synthesis of α-L-LNA analogues

The present invention may also be a method for the synthesis an α-L-LNAanalogue e.g. α-L-oxy-LNA, α-L-thio-LNA or α-L-amino-LNA of the generalformula VIII

wherein

-   X is selected from —CH₂—, —NR^(H)—, —O—, and —S—;-   Z is selected from —CH₂—, —NR^(H)—, —O—, —S—, and —Se—;-   B is a nucleobase;-   R³ is selected from —R^(H), —N₃, —NR^(H)R^(H)*, —NR^(H)C(O)R^(H)*,    —C(O)NR^(H)R^(H)*, —OR^(H), —OC(O)R^(H), —C(O)OR^(H), —SR^(H),    —SC(O)R^(N), and tri(C₁₋₆-alkyliaryl)silyloxy;

each R^(H) and R^(H)* independently being selected from hydrogen,optionally substituted C₁₋₆-alkyl, optionally substituted aryl, andoptionally substituted aryl-C₁₋₆-alkyl;

-   A⁴ and A^(s) independently are selected from C₁₋₆-alkylene; and-   R⁵ is selected from iodo, bromo, chloro, C₁₋₅-alkylsulfonyloxy    optionally substituted with one or more substituents selected from    halogen and phenyl optionally substituted with one or more    substituents selected from nitro, halogen and C₁₋₆-alkyl, and    arylsulfonyloxy optionally substituted with one or more substituents    selected from nitro, halogen, C₁₋₆-alkyl, and C₁₋₆-alkyl substituted    with one or more halogen;

said method comprising the following steps:

treating an intermediate of the general formula IX:

wherein

-   X, B, R³, A⁴, and A⁵ are as defined above;-   R² is selected from iodo, C₁₋₆-alkylsulfonyloxy optionally    substituted with one or more substituents selected from halogen and    phenyl optionally substituted with one or more substituents selected    from nitro, halogen and C₁₋₆-alkyl, and arylsulfonyloxy optionally    substituted with one or more substituents selected from nitro,    halogen, C₁₋₆-alkyl, and C₁₋₆-alkyl substituted with one or more    halogen;-   R³ and R² may together form an epoxide; and-   R⁴ and R⁵ independently are as defined for R⁵ above, or R⁴ and R⁵    together constitutes a tetra(C₁₋₆-alkyl)disiloxanylidene group;

with a nucleophile selected from halogen, ⁻N₃, ⁻NR^(H)R^(H)*, ⁻OR^(H),⁻OH, ⁻SR^(H), ⁻⁻S, ⁻SeR^(H), ⁻⁻Se, ⁻NR^(H)C(O)R^(H)*, ⁻SC(O)R^(H), andorganometallic hydrocarbyl radicals,

so as to substitute R², and

effecting ring-closure between the C2′ and C4′ positions so as to yieldthe LNA analogue of the formula VIII.

The interesting embodiment of intermediates of formula I wherein R² andR³ together forming an epoxide is particularly interesting in thesynthesis of α-L-oxy-LNA, α-L-thio-LNA or α-L-amino-LNA using a compoundof Formula IX, discussed infra.

In a further particularly interesting embodiment, the intermediate offormula IX has the formula X

wherein B, R³, R⁴ and R⁵ are as defined above.

The intermediate of Formula IX is reacted with a nucleophile selectedfrom halogen, ⁻N₃, ⁻NR^(H)R^(H)*, ⁻OR^(H), ⁻OH, ⁻SR^(H), ⁻⁻S,⁻NR^(H)C(O)R^(H)*, ⁻SC(O)R^(H), and organometallic hydrocarbyl radicals,so as to substitute R².

One particular advantage of using the common intermediate, X, in thisinvention in the reaction with hydroxide or an alkoxide such as3-hydroxylpropionitrile alkoxide as the nucleophile is that theα-L-structure is made in one-pot. Thus, substitution of the triflate byhydroxide or 3-hydroxylpropionitrile alkoxide produces an alcohol thatis immediately cyclised.

Embodiments relating to the synthesis of LNA analogues described supraare also applicable to the synthesis of α-L-LNA analogues.

The Novel Intermediates

It is believed that the majority of the intermediates (compounds ofFormula I) represent novel compounds, thus the present invention alsoprovides compounds of the formula I

wherein

-   X is selected from —CH₂—, —NR^(H)—, —O—, and —S—;-   B is a nucleobase;-   R² is selected from iodo, C₁₋₆-alkylsulfonyloxy optionally    substituted with one or more substituents selected from halogen and    phenyl optionally substituted with one or more substituents selected    from nitro, halogen and C₁₋₆-alkyl, and arylsulfonyloxy optionally    substituted with one or more substituents selected from nitro,    halogen, C₁₋₆-alkyl, and C₁₋₆-alkyl substituted with one or more    halogen;-   R³ is selected from —R^(H), —N₃, —NR^(H)R^(H)*, —NR^(H)C(O)R^(H)*,    —C(O)NR^(H)R^(H)*, —OR^(H), —OC(O)R^(H), —C(O)OR^(H), —SR^(H),    —SC(O)R^(H), and tri(C₁₋₆-alkyliaryl)silyloxy;-   R³ and R² may together form an epoxide;

each R^(H) and R^(H)* independently being selected from hydrogen,optionally substituted C₁₋₆-alkyl, optionally substituted aryl, andoptionally substituted aryl-C₁₋₆-alkyl;

-   A⁴ and A⁵ independently are selected from C₁₋₅-alkylene; and-   R⁴ and R⁵ independently are selected from iodo, bromo, chloro,    C₁₋₆-alkylsulfonyloxy optionally substituted with one or more    substituents selected from halogen and phenyl optionally substituted    with one or more substituents selected from nitro, halogen and    C₁₋₆-alkyl, and arylsulfonyloxy optionally substituted with one or    more substituents selected from nitro, halogen, C₁₋₆-alkyl, and    C₁₋₅-alkyl substituted with one or more halogen, or R⁴ and R⁵    together constitutes a tetra(C₁₋₆-alkyl)disiloxanylidene group;    with the proviso that the compound is not selected from-   1-(3-azido-3-deoxy-2,5-di-O-methanesulfonyl-4-C-(methansulfonyloxymethyl)-β-D-erythro-pentofuranosyl)thymine,-   1-(3-O-benzyl-2,5-di-O-methanesulfonyl-4-C-(methansulfonyloxymethyl)-β-D-erythro-pentofuranosyl)thymine,    and-   1-(3-O-benzyl-2,5-di-O-methanesulfonyl-4-C-(nnethansulfonyloxymethyl)-α-L-threo-pentofuranosyl)thymine.

Particular and preferred subgroups of the compounds of formula I are asdescribed above for the compound I under Synthesis of LNA analogues. Inparticular, particular subclasses of compounds have the formula II, inparticular and the formula III.

Examples of particularly interesting specific compounds are thoseillustrated in FIG. 13.

It is presently believed that a particularly interesting compound whichis particularly useful for the preparation of(1R,3R,4R,7S)-7-Benzyloxy-1-methansulfonyloxymethyl-3-(thymin-1-yl)-2-oxa-5-azabicyclo[2:2:1]heptane(33) and(1R,3R,4R,7S)-7-Benzyloxy-1-methansulfonyloxymethyl-3-(thymin-1-yl)-2-oxa-5-thiabicyclo[2:2:1]heptane(60) is1-(3-O-Benzyl-5-O-methanesulfonyl-4-C-methanesulfonyloxymethyl-2-O-trifluoromethanesulfonyl-β-D-threo-pentofuranosyl)thymine(31) (see FIG. 9).

Particular and preferred subgroups of the compounds of formula I aredescribed above under Synthesis of α-L-LNA analogues. In particular, aparticular subclass of compounds has the formula IX and particularlyformula X, and wherein R² and R³ together form an epoxide.

Preparation of the Novel Intermediates

The compounds (intermediates) of the formula I can be prepared byinversion of the orientation of the C2′ substituent in a similarcompound in which the C2′ substituent is a leaving group. Thus, thepresent invention also relates to a method for the synthesis of acompound of the formula I

wherein

-   X is selected from —CH₂—, —NR^(H)—, —O—, and —S—;-   B is a nucleobase;-   R² is selected from iodo, C₁₋₅-alkylsulfonyloxy optionally    substituted with one or more substituents selected from halogen and    phenyl optionally substituted with one or more substituents selected    from nitro, halogen and C₁₋₅-alkyl, and arylsulfonyloxy optionally    substituted with one or more substituents selected from nitro,    halogen, C₁₋₆-alkyl, and C₁₋₆-alkyl substituted with one or more    halogen;-   R³ is selected from —R^(H), —N₃, —NR^(H)R^(H)*, —NR^(H)C(O)R^(H)*,    —C(O)NR^(H)R^(H)*, —OR^(H), —OC(O)R^(H), —C(O)OR^(H), —SR^(H),    —SC(O)R^(N), and tri(C₁₋₆-alkyliaryl)silyloxy;

each R^(H) and R^(H)* independently being selected from hydrogen,optionally substituted C₁₋₆-alkyl, optionally substituted aryl, andoptionally substituted aryl-C₁₋₅-alkyl;

-   A⁴ and A⁵ independently are selected from C₁₋₆-alkylene; R³ and R²    may together form an epoxide and R⁴ and R⁵ independently are    selected from iodo, bromo, chloro, C₁₋₆-alkylsulfonyloxy optionally    substituted with one or more substituents selected from halogen and    phenyl optionally substituted with one or more substituents selected    from nitro, halogen and C₁₋₆-alkyl, and arylsulfonyloxy optionally    substituted with one or more substituents selected from nitro,    halogen, C₁₋₆-alkyl, and C₁₋₆-alkyl substituted with one or more    halogen.

said method comprising inversion of orientation of the substituent inthe C2′ position of a compound of the formula VII

wherein

-   R²* is a leaving group selected from iodo, C₁₋₆-alkylsulfonyloxy    optionally substituted with one or more substituents selected from    halogen and phenyl optionally substituted with one or more    substituents selected from nitro, halogen and C₁₋₆-alkyl, and    arylsulfonyloxy optionally substituted with one or more substituents    selected from nitro, halogen, C₁₋₆-alkyl, and C₁₋₆-alkyl substituted    with one or more halogen; and-   X, B, R³, R⁴, A⁴, R⁵ and A⁵ are as defined above.

Particular examples of R²* groups are iodo, methanesulfonyloxy,trifluoromethanesulfonyl-oxy, ethanesulfonyloxy,2,2,2-trifluoroethanesulfonyloxy, propanesulfonyloxy,iso-propanesulfonyloxy, butanesulfonyloxy, nonafluorobutanesulfonyloxy,pentanesulfonyloxy, cyclopentanesulfonyloxy, hexanesulfonyloxy,cyclohexanesulfonyloxy, α-toluenesulfonyl-oxy,2-chloro-α-toluenesulfonyloxy, ortho-toluenesulfonyloxy,meta-toluenesulfonyloxy, para-toluenesulfonyloxy, benzenesulfonyloxy,ortho-bromobenzenesulfonyloxy, meta-bromobenzenesulfonyloxy,para-bromobenzenesulfonyloxy, ortho-nitrobenzenesulfonyloxy,meta-nitrobenzenesulfonyloxy, and para-nitrobenzenesulfonyloxy, of whichtrifluoro-methylsulfonyloxy is a particularly preferred example.

Particular and preferred subgroups of the compounds of formula VIIcorresponds to those described above for the compound I under Synthesisof LNA analogues, mutatis mutantis. In particular, particular subclassesof compounds have the configuration corresponding to formula II,especially the configuration corresponding to formula III, except forthe orientation of the substituent on C2′.

In a particularly interesting embodiment of compounds of formula I, R³and R² together form an epoxide.

The novel compound illustrated in Formula I can be prepared by thegeneral route shown in FIG. 2.

The inversion of the orientation of the substituent on C2′ can effect invarious ways. If the nucleobase is a pyrimidine base, the inversion canbe facilitated by formation of a 2,2′-anhydro intermediate undersuitable conditions, e.g. use of a proton sponge e.g. DBU. Thetemperature is typically 0-100° C., such as 15-30° C., the reaction timeis typically 5 min to 24 hours, such as 1-6 hours, and the molar ratioof the base to the compound of the Formula VII is typically in the rangeof 5:1 to 1:1, such as in the range of 3:1 to 1:1. The polar aproticsolvent is typically DMF, THF, DMSO, or CH₃CN.

Although the above-mentioned method for the synthesis of the compound ofFormula I takes advantage of the 2,2′-anhydronucleoside construct, andtherefore only is applicable for nucleobases (such as pyrimidines) inwhich such a construct is possible, it should be understood that otherroutes will be similarly applicable for the inversion of orientation ofthe substituent in the C2′ position of a compound of the formula VII.

As an example, which is generally applicable for all nucleobases, andvery useful in the instances where the nucleobase is a purine typenucleobase, the inversion is effected by reaction of the compound of theformula VII with an oxygen nucleophile.

A more specific example of the convergent synthesis strategy for thesynthesis of an intermediate having a purine-type nucleobase isillustrated in FIG. 3. Compound 13 is base protected (14) after whichthe 2′-OAc is hydrolysed selective as described elsewhere herein (15).The liberated 2′-OH is triflated (16) and reacted with a suitable oxygennucleophile (e.g. an acetate, benzoate, etc.) to invert thestereochemistry (17). The resulting ester is then selective hydrolysedas described elsewhere herein and the 2′-OH now in the threoconfiguration (18). Compound 18 is a purine equivalent to compound 30which can subsequently be converted to 2′-O-mesylate, i.e. anintermediate of the formula I, following the route illustrated in FIG.6.

As a further alternative, inversion can also be effected by oxidation ofa compound of Formula VII where R²* is OH, followed by subsequentstereo- and regioselective reduction, e.g. as outlined in FIG. 4.

The starting materials of Formula VII for the method according to theinvention may be prepared as described in the literature (Koshkin, A.;Fensholdt, J.; Pfundheller, H. M.; Lomholt, C. J. Org. Chem. 2001. 66,8504-8512).

As a more specific example, the preferred general intermediate shown informula III can be prepared as shown below (FIG. 5). Thus,(2-O-acetyl-3-O-benzyl-4-C-methanesulfonyloxymethyl-5-O-methanesulfonyl-β-D-erythro-pentofuranosyl)-nucleobase)(23) is converted by a mild deacetylation for the liberation of the2′-hydroxy group to the compound (24) without the subsequent ringclosurethat affords the oxy-LNA skeleton. The 2′-hydroxy group is thenmesylated to afford(3-O-benzyl-4-C-methanesulfonyl-oxymethyl-2,5-O-dimethanesulfonyl-β-D-erythro-pentofuranosyl)-nucleobase)(25).

Thus, the present invention also relates to a method for the synthesisof a compound of the formula IX and X as described above under Synthesisof α-L-LNA analogues.

In view of the above, the present invention also provides method for thesynthesis of an LNA analogue of the formula IV

said method comprising synthesis of a compound of the formula I from acompound of the formula VII as defined in the method above, andconversion of the compound of the formula I to an LNA analogues of theformula IV as defined further above.Definitions

In the present context, the term “C₁₋₆-alkyl” means a linear, cyclic orbranched hydrocarbon group having 1 to 6 carbon atoms, such as methyl,ethyl, propyl, iso-propyl, butyl, tert-butyl, iso-butyl, pentyl,cyclopentyl, hexyl, cyclohexyl, in particular methyl, ethyl, propyl,iso-propyl, tert-butyl, iso-butyl and cyclohexyl.

The term “C₁₋₆-alkylene” is intended to mean a linear hydrocarbonbiradical having 1-6 carbon atoms, such as methylene, 1,2-ethylene,1,3-propylene, 1,2-propylene, 1,4-butylene, etc.

The term “optionally substituted” in connection with the terms“C₁₋₆-alkyl” and “C₁₋₆-alkylene” is intended to mean that the group inquestion may be substituted one or several times, preferably 1-3 times,with group(s) selected from hydroxy C₁₋₆-alkoxy (i.e. C₁₋₆-alkyl-oxy),carboxy, C₁₋₆-alkoxycarbonyl, C₁₋₆-alkylcarbonyl, aryl, aryloxycarbonyl,aryloxy, arylcarbonyl, amino, mono- and di(C₁₋₆-alkyl)amino; carbamoyl,mono- and di(C₁₋₆-alkyl)-aminocarbonyl, C₁₋₆-alkylcarbonylamino, cyano,carbamido, halogen, where any aryl may be substituted as specificallydescribe below for “optionally substituted aryl”.

In the present context the term “aryl” means a fully or partiallyaromatic carbocyclic ring or ring system, such as phenyl, naphthyl,1,2,3,4-tetrahydronaphthyl, anthracyl, and phenanthracyl, among whichphenyl is a preferred example.

The term “optionally substituted” in connection with the term “aryl” isintended to mean that the group in question may be substituted one orseveral times, in particular 1-3 times) with group(s) selected fromhydroxy, C₁₋₆-alkoxy, carboxy, C₁₋₆-alkoxy-carbonyl, C₁₋₆-alkylcarbonyl,aryl, amino, mono- and di(C₁₋₆-alkyl)amino, and halogen, wherein arylmay be substituted 1-3 times with C₁₋₄-alkyl, C₁₋₄-alkoxy, nitro, cyano,amino or halogen.

In the present context, the term “tri(C₁₋₆-alkyl/aryl)silyloxy” means asilyl group substituted with 0-3 C₁₋₆-alkyl groups and/or 0-3 arylgroups, with the provision that the total number of alkyl and arylgroups is 3. Illustrative examples are trimethylsilyloxy,allyldimethylsilyloxy, dimethylphenylsilyloxy, diphenylmethylsilyloxy,isopropyldimethylsilyloxy, tert-butyldimethylsilyloxy,tert-butyldiphenylsilyloxy, triethylsilyloxy, triisopropylsilyloxy,diethylisopropylsilyloxy, dimethylthexyl-isopropylsilyloxy,tribenzylsilyloxy, tri-para-xylylsilyloxy, triphenylsilyloxy,diphenylmethylsilyloxy, di-tert-butylmethylsilyloxy,tris(trimethylsilyloxy)silyloxy, tert-butylmethoxyphenylsilyloxy, andtert-butoxydiphenylsilyloxy.

In the present context, the term “tetra(C₁₋₆-alkyl)disiloxanylidene”means a —O—Si(C₁₋₆-alkyl)₂-O— biradical. A typical example is1,3-(1,1,3,3-tetraisopropyl)-disiloxanylidene.

“Halogen” includes fluoro, chloro, bromo, and iodo.

In the present context, the terms “nucleobase” covers naturallyoccurring nucleobases as well as non-naturally occurring nucleobases,i.e. heteroaromatic cyclic groups, e.g. monocyclic groups, bicyclicgroups, tricyclic groups, etc. It should be clear to the person skilledin the art that various nucleobases which previously have beenconsidered “non-naturally occurring” have subsequently been found innature. Thus, “nucleobase” includes not only the known purine andpyrimidine heterocycles, but also heterocyclic analogues and tautomersthereof. Illustrative examples of nucleobases are adenine, guanine,thymine, cytosine, uracil, purine, xanthine, diaminopurine,8-oxo-N⁶-methyladenine, 7-deazaxanthine, 7-deazaguanine,N⁴,N⁴-ethanocytosin, N⁶,N⁶-ethano-2,6-diaminopurine, 5-methylcytosine,5-(C³—C⁶)-alkynylcytosine, 5-fluorouracil, 5-bromouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine,isoguanin, inosine, N⁶-allylpurines, N⁶-acylpurines, N⁶-benzylpurine,N⁶-halopurine, N⁶-vinylpurine, N⁶-acetylenic purine, N⁶-acyl purine,N⁶-hydroxyalkyl purine, N⁶-thioalkyl purine, N²-alkylpurines,N⁴-alkylpyrimidines, N⁴-acylpyrimidines, N⁴-benzylpurine,N⁴-halopyrimidines, N⁴-vinylpyrimidines, N⁴-acetylenic pyrimidines,N⁴-acyl pyrimidines, N⁴-hydroxyalkyl pyrimidines, N⁶-thioalkylpyrimidines, 6-azapyrimidine, including 6-azacytosine, 2- and/or4-mercaptopyrimidine, uracil, C⁵-alkylpyrimidines, C⁵-benzylpyrimidines,C5-halopyrimidines, C⁵-vinylpyrimidine, C⁵-acetylenic pyrimidine,C⁵-acyl pyrimidine, C⁵-hydroxyalkyl purine, C⁵-amidopyrimidine,C⁵-cyanopyrimidine, C⁵-nitropyrimidine, C⁵-aminopyrimdine,N²-alkylpurines, N²-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl,trazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl, andpyrazolopyrimidinyl. Functional oxygen and nitrogen groups on the basecan be protected and deprotected if necessary or desirable. Suitableprotecting groups are well known to those skilled in the art, andincluded trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, andt-butyldiphenylsilyl, trityl, alkyl groups, acyl groups such as acetyland propionyl, methanesulfonyl, and p-toluenesulfonyl. Preferred basesinclude adenine, guanine, 2,6-diaminopurine, thymine, 2-thiothymine,cytosine, methyl cytosine, uracil, 5-fluorocytosine, xanthine,6-aminopurine, 2-aminopurine, 6-chloro-2-amino-purine, and6-chloropurine. Especially interesting nucleobases are adenine, guanine,thymine, cytosine, and uracil, which are considered as the naturallyoccurring nucleobases in relation to therapeutic and diagnosticapplication in humans.

EXAMPLES

For reactions conducted under anhydrous conditions glassware was driedovernight in an oven at 150° C. and was allowed to cool in a dessicatorover anhydrous KOH. Anhydrous reactions were carried out under anatmosphere of argon. Solvents were HPLC grade, of which DMF, pyridine,acetonitrile and dichloromethane were dried over molecular sieves (4 Åfrom Grace Davison) and THF was freshly destilled from Na•benzophenoneto a water content below 20 ppm. TLC was run on Merck silica 60 F₂₅₄aluminum sheets. Dry Column Vacuum Chromatography (DCVC) was performedaccording to the published procedure. ¹H, ¹³C, ¹⁹F, and ³¹P NMR spectrawere recorded at respectively 400 MHz, 100 MHz, 376 MHz, and 121 MHzwith solvents as internal standard (δ_(H): CDCl₃ 7.26 ppm, DMSO-d₆ 2.50;δ_(C): CDCl₃ 77.0 ppm, DMSO-d₆ 39.4 ppm). ³¹P NMR was run with 85% H₃PO₄as external standard. J values are given in Hz. Assignments of NMRspectra are based on 2D spectra and follow the standardcarbohydrate/nucleoside nomenclature (the carbon atom of the4′-C-substituent is numbered C1″) even though the systematic compoundnames of the bicyclic nucleoside derivatives are given according to thevon Baeyer nomenclature. Crude compounds were used without furtherpurification if they were ≧95% pure by TLC and HPLC-MS (RP C18 column,UV detection). Elemental analyses were obtained from the University ofCopenhagen, Microanalytical Department.

1-(2,5-Di-O-acetyl-4-C-acetyloxymethyl-3-O-benzyl-β-D-erythro-pentofuranosyl)thymine

To a stirred solution of3-O-benzyl-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-erythro-pentofuranose1 (Youssefyeh, R. D.; Verheyden, J. P. H.; Moffatt, J. G. J. Org. Chem.1979, 44, 1301-1309). (200 mg, 0.64 mmol) in acetic acid (3.69 mL, 64.4mmol) at 0° C. was added acetic anhydride (0.61 mL, 6.44 mmol) and concdH₂SO₄ (0.34 μL, 6.44 μmol). After 25 min the reaction mixture wasallowed to warm to rt. Stirring was continued for 2 h after which themixture was poured into ice cooled sat. aq NaHCO₃ (150 mL). The solutionwas extracted with dichloromethane (2×150 mL), and the combined organicphases were washed with sat. aq NaHCO₃ (2×100 mL), dried (Na₂SO₄),filtered and evaporated to dryness in vacuo to give the crude anomericmixture of the acetylated glycoside donor as a colorless liquid (258 mg,0.59 mmol). The liquid (246 mg, 0.56 mmol) was dissolved in anhydacetonitrile (5 mL) with stirring. Thymine (144 mg, 1.14 mmol) andN,O-bis(trimethylsilyl)acetamide (0.99 mL, 4.00 mmol) were added, andthe mixture was heated to reflux for 1.5 h and then cooled to 0° C.Trimethylsilyl triflate (0.23 mL, 1.25 mmol) was added dropwise during 5min and the mixture was heated to 80° C. for 3.5 h. The reaction mixturewas allowed to cool to rt, and ice cooled sat. aq NaHCO₃ (10 mL) wasadded. Extraction was performed with dichloromethane (2×20 mL), and thecombined organic phases were washed successively with sat. aq NaHCO₃(2×20 mL) and brine (20 mL), dried (Na₂SO₄), filtered and evaporated todryness in vacuo. The residue was purified by DCVC (0-1% MeOH indichloromethane v/v) to give the nucleoside (259 mg, 91%) as a whitesolid material. FAB-MS m/z found 505.0 ([MH]⁺, calcd 505.2); ¹H NMR(CDCl₃) δ9.93 (s, 1H, NH), 7.37-7.28 (m, 5H, Ph), 7.09 (d, J=0.9, 1H,H6), 5.79 (d, J=3.5, 1H, H1′), 5.53 (dd, J=6.3, 3.7, 1H, H2′), 4.64-4.08(m, 7H, CH ₂Ph, H3′, H5′a, H5′ b, H1″a, H1″b), 2.11 (s, 3H, CH₃C(O)),2.10 (s, 3H, CH₃C(O)), 2.07 (s, 3H, CH₃C(O)), 1.91 (s, 3H, CH₃); ¹³C NMR(CDCl₃) δ 170.4, 169.9, 163.9, 149.9 (CH₃ C(O), C2, C4), 137.1, 136.8,128.3, 128.0, 127.8 (C6, Ph), 111.0 (C5), 90.6 (C1′), 84.2 (C4′), 77.0(C3′), 74.2 (CH₂Ph), 73.7 (C2′), 63.6, 62.2 (C5′, C1″), 20.6, 20.5 (CH₃C(O)), 12.3 (CH₃).

1-(3-O-Benzyl-4-C-hydroxymethyl-β-D-erythro-pentofuranosypthymine

Nucleoside1-(2,5-Di-O-acetyl-4-C-acetyloxymethyl-3-O-benzyl-β-D-erythro-pentofuranosyl)thymine(149 mg, 0.30 mmol) was dissolved in a sat. solution of NH₃ in MeOH (15mL). The mixture was stirred overnight at rt in a sealed flask andevaporated to dryness under reduced pressure. The residue was dissolvedin EtOAc (30 mL) and washed with water (10 mL). The aq phase wasextracted with EtOAc (30 mL) and the combined organic phases werecoevaporated to dryness with acetonitrile (2×10 mL) under reducedpressure. The residue was purified by DCVC (1-4% MeOH in dichloromethanev/v), affording the nucleoside (93 mg, 84%) as a viscous liquid. R_(f)0.32 (10% MeOH in EtOAc, v/v); FAB-MS m/z found 379.0 ([MH]⁺, calcd379.1); ¹H NMR (DMSO-d₆) δ 11.29 (br s, 1H, NH), 7.73 (d, J=1.3, 1H,H6), 7.40-7.26 (m, 5H, Ph), 5.90 (d, J=6.2, 1H, H1′), 5.51 (d, J=7.5,1H, OH), 5.18 (t, J=5.0, 1H, OH), 4.86 (t, J=5.49, 1H, OH), 4.81 (d,3=11.7, 1H), 4.56 (d, J=11.7, 1H), 4.36 (q, J=6.3, 1H, H2′), 4.08 (d,J=5.5, 1H, H3′), 3.60-3.50 (m, 4H) (H5′, H1″, CH₂Ph), 1.79 (d, J=1.1,3H, CH₃); ¹³C NMR (DMSO-d₆) δ163.6 (C4), 150.7 (C2), 138.6, 136.3,128.0, 127.2 (C6, Ph), 109.3 (C5), 87.7, 87.5 (C1′, C4′), 78.5 (C3′),73.3 (C2′), 72.7, 62.8, 61.3 (C5′, C1″, CH₂Ph), 12.2 (CH₃); Anal. calcdfor C₁₈H₂₂N₂O₇.0.25 H₂O: C, 56.5; H, 5.9; N, 7.3. Found: C, 56.5; H,5.9; N, 7.0.

1-(3-O-Benzyl-2,5-di-O-methanesulfonyl-4-C-(methanesulfonyloxymethyl)-β-D-erythro-pentofuranosyl)thymine(28)

Nucleoside1-(3-O-Benzyl-4-C-hydroxymethyl-β-D-erythro-pentofuranosyl)thymine (0.83g, 3.2 mmol) was dissolved in anhyd pyridine (20 mL) and cooled to 0° C.with stirring. Methanesulfonyl chloride (0.85 mL, 11 mmol) was addeddropwise and the reaction was allowed to reach 15° C. over 3 h. Thereaction was quenched with sat. aq NaHCO₃ (50 mL) and transferred to aseparatory funnel with brine (50 mL) and EtOAc (100 mL). The phases wereseparated and the aq phase extracted with EtOAc (2×50 mL). The combinedorganic phases were extracted with brine (100 mL), dried (Na₂SO₄),filtered and evaporated in vacuo to give a viscous yellow liquid. Theliquid was dissolved in a mixture of dichloromethane and toluene andevaporated in vacuo to give nucleoside 28 (1.48 g, 93%) as a white foam.Analytical data were identical to those previously published.(Håkansson, A. E.; Koshkin, A.; Sørensen, M. D.; Wengel, J. J. Org.Chem. 2000, 65, 5161-5166.)

1-(3-O-Benzyl-5-O-methanesulfonyl-4-C-methanesulfonyloxymethyl-β-D-erythro-pentofuranosyl)thymine(27)

Nucleoside 26 (Koshkin et al., J. Org. Chem. 2001, 66, 8504-8512) (30 g,52 mmol) was dissolved in MeOH (600 mL), and the solution was cooled to0° C. Freshly prepared sat. methanolic ammonia (600 mL) was added, andthe mixture was allowed to reach rt. After 5 h at rt the reaction wasquenched with glacial acetic acid (50 mL) and transferred to a beaker,where it was neutralised with sat. aq NaHCO₃. EtOAc (900 mL) and brine(500 mL) was added and the phases were separated. The aq phase wasextracted with EtOAc (3×500 mL) and the combined organic phases werewashed with sat. aq NaHCO₃ (500 mL) and brine (500 mL). The organicphase was dried (Na₂SO₄), filtered and the solvent removed in vacuo toafford 27 (27 g, 97%) as a white foam. R_(f)=0.33 (100% EtOAc); ESI-MSm/z found 557.0 ([MNa]⁺. Calcd 557.1); ¹FI NMR (CDCl₃) δ 10.21 (br s,1H, NH), 7.33-7.25 (m, 6H, Ph, H6), 5.77 (d, J=3.9, 1H, H1′), 4.84 (d,J=11.4, 1H, H3′), 4.59-4.57 (m, 3H), 4.42-4.37 (m, 3H), 4.26-4.19 (m,2H) (H2′, H2″, H5″, CH ₂Ph, OH), 2.98 (s, 3H, CH₃), 2.76 (s, 3H, CH₃),1.80 (s, 3H, CH₃); ¹³C NMR (CDCl₃) δ162.5 (C4), 151.0 (C2), 136.7 (Ph),136.2 (C6), 128.5, 128.3, 128.2 (Ph), 111.3 (C5), 92.1 (C1′), 84.0(C4′), 77.7 (C3′), 74.1, 73.5 (C2′, CH₂Ph), 68.6, 68.3 (C5′, C1″), 37.2,37.1 (Ms), 12.0 (CH₃); Anal. calcd for C₂₀H₂₆N₂O₁₁S₂: C, 44.9; H, 4.9;N, 5.2. Found: C, 45.0; H, 4.7; N, 5.1.

1-(3-O-Benzyl-2,5-di-O-methanesulfonyl-4-C-(methanesulfonyloxymethyl)-β-D-erythro-pentofuranosyl)thymine(28)

Nucleoside 27 (20 g, 37 mmol) was dissolved in anhyd dichloromethane(100 mL) and anhyd pyridine (100 mL) was added. The solution was cooledto 0° C. and methanesulfonyl chloride (4.4 mL, 56 mmol) was addeddropwise. After 2 h the reaction was quenched with sat. aq NaHCO₃ (200mL), and the phases were separated. The aq phase was extracted withdichloromethane (2×150 mL), and the combined organic phases were washedwith aq HCl (1 M, 2×200 mL), sat. aq NaHCO₃ (2×250 mL) and brine (250mL). The organic phase was dried (Na₂SO₄), filtered and the solvent wasremoved in vacuo. The crude product was co-evaporated with tolueneaffording 28 (22 g, 96%) as a white foam. R_(f)=0.41 (100% EtOAc);ESI-MS m/z found 635.0 ([MNa]⁺, calcd 635.1). All analytical data wereidentical to those previously reported. (Håkansson, A. E.; Koshkin, A.;Sørensen, M. D.; Wengel, J. J. Org. Chem. 2000, 65, 5161-5166)

2,2′-Anhydro-1-(3-O-benzyl-5-O-methanesulfonyl-4-C-methanesulfonyloxymethyl-β-D-threo-pentofuranosypthymine(29)

Nucleoside 28 (10 g, 16.3 mmol) was dissolved in anhyd acetonitrile (100mL) and DBU (2.69 mL, 18.0 mmol) was added. The product slowlyprecipitated from the reaction mixture. After 2 h the reaction wascompleted and concentrated in vacuo to facilitate precipitation. Thereaction mixture was cooled to −20° C. and the product collected byfiltration to afford nucleoside 29 (7.64 g, 91%) as a white solidmaterial. FAB-MS m/z found 517.0 ([MH]⁺, calcd 517.1); ¹H NMR (DMSO-d₆)δ7.79 (d, J=1.3, 1H, H6), 7.45-7.32 (m, 5H, Ph), 6.40 (d, J=6.0, 1H,H1′), 5.60 (dd, J=6.1, 2.8, 1H, H2′), 4.82 (d, J=11.5, 1H, CH ₂Ph), 4.70(d, J=11.5, 1H, CH ₂Ph), 4.51 (d, J=2.8, 1H, H3′), 4.43 (d, J=10.6, 1H),4.36 (d, J=6.2, 1H), 4.33 (d, J=5.9, 1H), 4.25 (d, J=11.0, 1H) (H5′,H1″), 3.22 (s, 3H, Ms), 3.16 (s, 3H, Ms), 1.80 (5, J=1.1, 3H, CH₃); ¹³CNMR (DMSO-d₆) δ 171.5 (C4), 159.1 (C2), 136.9, 132.1, 128.5, 128.1,127.9 (C6, Ph), 117.1 (C5), 89.1 (C1′), 86.1 (C2′), 85.4 (C4′), 83.7(C3′), 72.4 (CH ₂Ph), 68.6, 68.0 (C5′, C1″), 36.9, 36.8 (Ms), 13.6(CH₃); Anal. calcd for C₂₀H₂₄N₂O₁₀S₂: C, 46.5; H, 4.7; N, 5.4. Found: C,46.6; H, 4.8; N, 5.3.

1-(3-O-Benzyl-5-O-methanesulfonyl-4-C-methanesulfonyloxymethyl-β-D-threo-pentofuranosyl)thymine(30)

Nucleoside 29 (3.70 g, 7.16 mmol) was suspended in a mixture of acetone(160 mL) and aq H₂SO₄ (0.1 M, 160 mL). The mixture was heated to refluxovernight with stirring. After cooling to rt a white solid precipitated.The volume was reduced to approx. ½ in vacuo and a white solid wasisolated by filtration. The solid was washed thoroughly with water anddried in vacuo to give nucleoside 30 (3.77 g, 98%) as a white solid.FAB-MS m/z found 535.0 ([MH]⁺, calcd 535.1); ¹H NMR (DMSO-d₆) δ11.35 (s,1H, NH), 7.41-7.32 (m, 6H, H6, Ph), 6.20 (d, J=5.0, 1H, H1′), 6.10 (d,J=4.8, 1H, 2′-OH), 4.77 (d, J=11.9, 1H, CH ₂Ph), 4.67 (d, J=11.9, 1H, CH₂Ph), 4.56 (d, J=10.6, 1H), 4.50-4.41 (m, 3H), 4.32 (d, J=10.6, 1H),4.16 (d, J=3.7, 1H, H3′), 3.25 (s, 3H, Ms), 3.20 (s, 3H, Ms), 1.79 (s,3H, CH₃); ¹³C NMR (DMSO-d₆) δ 163.9 (C4), 150.6 (C2), 137.8, 137.6,128.4, 127.9, 127.7 (C6, Ph), 108.2 (C5), 84.8 (C1′), 84.3 (C3′), 81.7(C4′), 73.3 (C2′), 72.3 (CH₂Ph), 68.1, 67.6 (C5′, C1″), 37.0, 36.8 (Ms),12.2 (CH₃); Anal. calcd for C₂₀H₂₆N₂O₁₁S₂: C, 44.9; H, 4.9; N, 5.2.Found: C, 44.5; H, 4.8; N, 5.1.

1-(3-O-Benzyl-5-O-methanesulfonyl-4-C-methanesulfonyloxymethyl-2-O-trifluoromethanesulfonyl-β-D-threo-pentofuranosyl)thymine(31)

Nucleoside 30 (300 mg, 0.56 mmol) was dissolved in anhyd pyridine (2×5mL) and concentrated in vacuo to remove water traces. The compound wasdissolved in a mixture of anhyd dichloromethane (20 mL) and anhydpyridine (0.45 mL, 5.60 mmol) followed by the addition of DMAP (274 mg,2.24 mmol). After cooling to 0° C. trifluoromethanesulfonic anhydride(0.19 mL, 1.12 mmol) was added dropwise during 30 min. The reactionmixture was stirred for an additional 1.5 h and poured into ice cooledsat. aq NaHCO₃ (20 mL). The organic phase was separated and washedsuccessively with aq HCl (1 M, 2×20 mL) and sat. aq NaHCO₃ (2×20 mL),dried (Na₂SO₄), filtered and evaporated in vacuo. The residue waspurified by DCVC (0-100% EtOAc in n-heptane v/v) yielding nucleoside 31(302 mg, 80%) as a white foam. FAB-MS m/z found 667.0 ([MH]⁺, calcd667.0); ¹H NMR (DMSO-d₆) δ 11.62 (br s, 1H, NH), 7.51 (s, 1H, H6),7.40-7.33 (m, 5H, Ph), 6.45 (br s, 1H, H1′), 5.91 (t, J=6.0, 1H, H2′),4.97 (d, J=5.7, 1H, H3′), 4.82-4.36 (m, 6H, CH ₂Ph, H5′a, H5′ b, H1″a,H1″b), 3.30 (s, 3H, Ms), 3.24 (s, 3H, Ms), 1.81 (s, 3H, CH₃); ¹³C NMR(DMSO-d₆) δ 163.3 (C4), 150.0 (C2), 136.5, 128.3, 128.0, 127.8 (C6, Ph),117.6 (q, J=320, CF₃), 110.1 (C5), 88.0 (C1′), 81.7, 81.0 (C3′, C4′),73.1 (CH₂Ph), 68.0, 67.6 (C5′, C1″), 36.7, 36.6 (Ms), 11.8 (CH₃); Anal.calcd for C₂₁H₂₅F₃N₂O₁₃S₃: C, 37.8; H, 3.8; N, 4.2. Found: C, 38.1; H,3.8; N, 4.1.

1-(2-Azido-3-O-benzyl-2-deoxy-5-O-methanesulfonyl-4-C-(methanesulfonyloxymethyl)-β-D-erythro-pentofuranosyl)thymine(32)

Method A: To a solution of nucleoside 31 (215 mg, 0.32 mmol) in anhydDMF (10 mL) NaN₃ (23 mg, 0.35 mmol) and 15-crown-5 (64 μL, 0.32 mmol)was added. The mixture was stirred at 80° C. for 1 h and then cooled tort whereupon water (20 mL) was added. The solution was extracted withEtOAc (50 mL) and the organic phase was washed with sat. aq NaHCO₃ (2×20mL), dried (Na₂SO₄), filtered and evaporated to dryness in vacuo. Theresidue was purified by DCVC (50-100% EtOAc in n-heptane v/v) yieldingnucleoside 32 (164 mg, 91% from 31) as a white foam. Analytical datawere identical to those reported above.

Method B: A solution of nucleoside 30 (5.35 g, 10 mmol) in anhyddichloromethane (300 mL) was cooled to 0° C. Anhyd pyridine (8.08 mL,100 mmol) and DMAP (4.89 g, 40 mmol) was added followed by the dropwiseaddition of trifluoromethansulfonic anhydride (3.3 mL, 20 mmol). After 2h at 0° C. the reaction was quenched by the addition of ice cold sat. aqNaHCO₃ (200 mL) and the reaction mixture was transferred to a separatoryfunnel. The phases were separated and the aq phase was extracted withdichloromethane (200 mL). The combined organic phases were washed withaq HCl (1.0 M, 2×300 mL) and sat. aq NaHCO₃ (300 mL), dried (Na₂SO₄),filtered and concentrated in vacuo to give a white solid. The solid wasdissolved in anhyd DMF (300 mL) and NaN₃ (1.86 g, 30 mmol) was added.After stirring at rt for 4 h brine (300 mL) was added and the mixturewas transferred to a separatory funnel. The aq phase was extracted withdichloromethane (3×200 mL) and the combined organic phases were dried(Na₂SO₄), filtered and concentrated in vacuo yielding a yellow residuethat was purified by DCVC (Ø 5 cm, 25-100% EtOAc in n-heptane v/v, 5%increments, 100 mL fractions) affording nucleoside 32 (5.1 g, 91% from30) as a white solid. Analytical data were identical to those reportedabove.

(1R,3R,4R,7S)-7-Benzyloxy-1-methansulfonyloxymethyl-3-(thymin-1-yl)-2-oxa-5-azabicyclo[2:2:1]heptane(33).

To a solution of 32 (5.83 g, 10.4 mmol) in THF (300 mL) at rt aq NaOH(2.0 M, 104 mL, 208 mmol) and PMe₃ in THF (1.0 M, 20.8 mL, 20.8 mmol)was added with stirring. After 8 h the THF was partly removed underreduced pressure. Brine (200 mL) and EtOAc (300 mL) was added and thephases were separated. The aq phase was extracted with EtOAc (2×300 mL)and dichloromethane (2×300 mL). The combined organic phases were dried(Na₂SO₄), filtered and concentrated in vacuo to give nucleoside 33 (4.22g, 93%) as a white solid. R_(f)=0.15 (10% MeOH in EtOAc, v/v); ESI-MSm/z found 438.0 ([MH]⁺, calcd 438.1); ¹H NMR (DMSO-d₆) δ11.33 (br s, 1H,NH), 7.46 (s, 1H, H6), 7.36-7.27 (m, 5H, Ph), 5.44 (s, 1H, H1′), 4.67(d, J=11.7, 1H), 4.59 (d, J=11.5, 1H), 4.56 (d, J=11.9, 1H), 4.52 (d,J=11.7, 1H) (H5′, CH ₂Ph), 3.84 (s, 1H, H3′), 3.65 (s, 1H, H2′), 3.26(s, 3H, Ms), 3.06 (d, J=10.1, 1H, H1″a), 2.78 (d, J=9.9, 1H, H1″b), 1.77(s, 3H, CH₃); ¹³C NMR (DMSO-d₆) δ 163.9 (C4), 150.1 (C2), 137.9, 134.7,128.2, 127.7, 127.6 (C6, Ph), 108.3 (C5), 88.4 (C1′), 85.6 (C4′), 76.3(C3′), 70.9, 66.6 (CH₂Ph, C5′), 59.4 (C2′), 50.1 (C1″), 36.9 (Ms), 12.3(CH₃); Anal. calcd for C₁₉H₂₃N₃O₇S: C, 52.1; H, 5.3; N, 9.6. Found: C,52.0; H, 5.2; N, 9.2.

(1R,3R,4R,7S)-7-Benzyloxy-1-methansulfonyloxymethyl-5-methyl-3-(thymin-1-yl)-2-oxa-5-azabicyclo[2:2:1]heptane(34).

To a solution of 33 (4.22 g, 9.64 mmol) in formic acid (20 mL)formaldehyde (37% aq solution, 20 mL) was added with stirring and thereaction mixture was heated to 80° C. After 1 h the reaction was dilutedwith EtOAc (150 mL) and quenched by carefully pouring it into sat. aqNaHCO₃ (100 mL). The phases were separated and the organic phase waswashed with sat. aq NaHCO₃ (4×100 mL). The combined aq phases wereextracted with dichloromethane (2×200 mL). The combined organic phaseswere dried (Na₂SO₄), filtered and concentrated under reduced pressure.Purification by DCVC (Ø 6 cm, 0-15% MeOH in EtOAc v/v, 1% increments,100 mL fractions) afforded nucleoside 34 (3.89 g, 90%) as an off-whitesolid. R_(f)=0.30 (10% MeOH in EtOAc, v/v); ESI-MS m/z found 452.1([MH]⁺, calcd 452.1); ¹H NMR (DMSO-d₆) 11.34 (br s, 1H, NH), 7.43 (s,1H, H6), 7.34-7.28 (m, 5H, Ph), 5.58 (s, 1H, H1′), 4.67 (m, 4H, H5′,CH₂Ph), 3.88 (s, 1H, H3′), 3.58 (s, 1H, H2′), 3.27 (s, 3H, Ms), 2.98 (d,J=9.7, 1H, H1″a), 2.76 (d, J=9.7, 1H, H1″b), 2.57 (s, 3H, NCH₃), 1.76(s, 3H, CH₃); ¹³C NMR (DMSO-d₆) δ 163.9 (C4), 149.9 (C2), 137.6 (Ph),134.6 (C6), 128.3, 127.7 (Ph), 108.4 (C5), 86.1 (C1′), 85.3 (C4′), 77.3(C3′), 71.0, 66.3 (CH₂Ph, C5′), 64.9 (C2′), 58.7 (C1″), 40.8 (NCH₃),36.9 (Ms), 12.3 (CH₃); Anal. calcd for C₂₀H₂₅N₃O₇S.0.25 H₂O: C, 52.7; H,5.6; N, 9.1. Found: C, 52.9; H, 5.6; N, 8.9.

(1R,3R,4R,7S)-7-Benzyloxy-1-hydroxymethyl-5-methyl-3-(thymin-1-yl)-2-oxa-5-azabicyclo[2:2:1]heptane(35).

Compound 34 (3.00 g, 6.64 mmol) was dissolved in anhyd DMF (30 mL) andsodium benzoate (1.93 g, 13.3 mmol) was added. The reaction mixture washeated to 100° C. for 7 h and then cooled to rt. Sodium methoxide (1.44g, 26.6 mmol) was added and after 1 h the reaction was diluted withdichloromethane (100 mL) and washed with brine (2×100 mL). The combinedaq phases were extracted with dichloromethane (2×50 mL). The combinedorganic phases were dried (Na₂SO₄) and concentrated under reducedpressure. The residue was dissolved in aq HCl (1 M, 15 mL) andlyophilised yielding an off-white solid. Purification by DCVC (Ø 4 cm,0-10% MeOH in dichloromethane v/v, 0.5% increments, 50 mL fractions)afforded the hydrochloride salt of nucleoside 35 (2.72 g, 98%) as anoff-white solid. R_(f)=0.19 (7% MeOH in dichloromethane, v/v); ESI-MSm/z found 374.1 ([MH]⁺, calcd 374.2), 408.1, 410.1 ([MCl]⁻, calcd 408.1,410.1); ¹H-NMR (DMSO-d₆) δ11.43 (br s, 1H, NH), 7.63 (s, 1H, H6),7.45-7.29 (m, 5H, Ph), 5.60 (s, 1H, H1′), 4.80 (t, J=5.7, 1H, 5′-OH),4.67-4.50 (m, 2H, CH ₂Ph), 3.87 (s, 1H, H3′), 3.67 (d, J=6.0, 2H, H5′),3.38 (s, 1H, H2′), 2.88 (d, J=9.2, 1H, H1″a), 2.66 (d, J=9.5, 1H, H1″b),2.57 (s, 3H, NCH₃), 1.75 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆) δ 164.0 (C4),149.8 (C2), 137.0 (Ph), 134.4 (C6), 128.5, 127.8 (Ph), 108.9 (C5), 88.4(C1′), 88.0 (C4′), 77.8 (C3′), 71.0, (CH₂Ph), 66.0, 65.7 (C2′, C5′),61.4 (C1″), 40.1 (NCH₃), 12.6 (CH₃); Anal. calcd for C₁₉H₂₃N₃O₅.HCl.H₂O:C, 53.3; H, 6.1; N, 9.8. Found: C, 53.0; H, 6.3; N, 9.6.

(1R,3R,4R,7S)-7-Hydroxy-1-hydroxymethyl-5-methyl-3-(thymin-1-yl)-2-oxa-5-azabicyclo[2:2:1]heptane(36).

Compound 35 (2.60 g, 6.64 mmol) was dissolved in glacial acetic acid (50mL) and the reaction flask was evacuated and filled with argon severaltimes. Pd(OH)₂ on charcoal (20% moist, 200 mg) was added and thereaction flask was evacuated and filled with hydrogen gas several times.The reaction was stirred vigorously under an atmosphere of hydrogen gasfor 8 h. The catalyst was removed by filtration through a plug ofcelite. The celite was washed thoroughly with hot methanol (200 mL). Thesolvents were removed in vacuo. The residue was dissolved in water (10mL) and lyophilised yielding the acetate salt of nucleoside 36 (2.10 g,97%) as off-white flakes. R_(f)=0.11 (0.5% Et₃N, 10% MeOH, 89.5% EtOAc,v/v/v); ESI-MS m/z found 284.1 ([MH]⁺, calcd 284.1). All analytical datawere identical to those previously reported.⁷

(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-5-methyl-3-(thymin-1-yl)-2-oxa-5-azabicyclo[2:2:1]heptane(37).

Compound 36 (2.00 g, 5.83 mmol) was dissolved in anhyd pyridine (2×50mL) and concentrated in vacuo. The nucleoside was dissolved in anhydpyridine (50 mL) and 4,4′-dimethoxytrityl chloride (2.96 g, 8.74 mmol)was added and the reaction was stirred at rt for 9 h. The reaction wasconcentrated to ½ volume in vacuo and the residue was diluted with EtOAc(100 mL). The organic phase was washed with sat. aq NaHCO₃ (3×100 mL)and brine (100 mL), dried (Na₂SO₄), filtered and concentrated underreduced pressure. Purification by DCVC (Ø 4 cm, 0-10% MeOH in EtOAc+0.5%TEA v/v, 0.5% increments, 50 mL fractions) afforded nucleoside 37 (3.13g, 92%) as off-white solid. R_(f)=0.38 (0.5% Et₃N, 10% MeOH, 89.5%EtOAc, v/v/v); ESI-MS m/z found 586.2 ([MH]⁺, calcd 586.2). Allanalytical data were identical to those previously reported. (Singh, S.K.; Kumar, R.; Wengel, J. J. Org. Chem. 1998, 63, 10035-10039)

(1R,3R,4R,7S)-7-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4′-dimethoxytrityloxymethyl)-5-methyl-3-(thymin-1-yl)-2-oxa-5-azabicyclo[2.2.1]heptane(38)

Compound 37 (500 mg, 0.85 mmol) was dissolved in anhyd dichloromethane(4 mL) and 4,5-dicyanoimidazole in MeCN (1.0 M, 0.59 mL, 0.59 mmol) wasadded at ambient temperature with stirring.2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.27 mL, 0.85mmol) was added dropwise to the reaction mixture. After 2 h the reactionwas diluted with dichloromethane (10 mL) and transferred to a separatoryfunnel and extracted with sat. aq NaHCO₃ (2×15 mL) and brine (15 mL).The combined aq phases were extracted with dichloromethane (10 mL). Theorganic phases were pooled and dried (Na₂SO₄). After filtration theorganic phase was evaporated in vacuo to give nucleoside 29 as aslightly yellow foam (660 mg, 98% yield). R_(f)=0.56 (0.5% Et₃N, 10%MeOH, 89.5% EtOAc, v/v/v); ESI-MS m/z found 786.3 ([MH]⁺, calcd 786.4).¹⁹P NMR (CDCl₃) δ149.8, 149.6. (Singh, S. K.; Kumar, R.; Wengel, J. J.Org. Chem. 1998, 63, 10035-10039)

(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-5-N-methyl-3-(4-N-benzoyl-5-methyl-cytosine-1-yl)-2-oxa-5-azabicyclo[2:2:1]heptane(43)

Compound 37 (1.5 g, 2.5 mmol) was dissolved in anhyd pyridine (25 mL).Acetic anhydride (2.4 mL, 25 mmol) was added and the reaction stirredfor 24 h at ambient temperature. The reaction was quenched with water(25 mL) and extracted with EtOAc (2×25 ml). The combined organic phaseswere washed with sat. aq. NaHCO₃ (2×50 ml), brine (50 ml), and dried(Na₂SO₄). The organic phase was filtered and evaporated in vacuo to givecompound 39 as a white foam. Residual water was removed from the crudeproduct by evaporation from anhyd MeCN. The product was then dissolvedin anhyd MeCN (50 ml) and Et₃N (3.5 mL, 25.3 mmol) was added followed by1,2,4-triazole (1.75 g, 25 mmol). The reaction mixture was cooled on anicebath and POCl₃ (0.48 mL, 5.0 mmol) was added dropwise to give a whiteslurry. After 15 min the reaction mixture was allowed to reach roomtemperature. The resulting yellow slurry was stirred under argon atambient temperature. After 4.5 h the reaction mixture was poured into aslurry of sat. aq NaHCO₃ (50 mL) and ice and extracted with EtOAc (3×25mL). The combined organic phases were washed with brine (100 ml) anddried (Na₂SO₄). Filtration and evaporation in vacuo afforded thetrialzolide 40 as a pink foam which was immediately dissolved in anhydMeCN (50 ml) and sat. aq NH₄OH (50 mL) was added. After stirring for 16h solid NaCl was added until the phases separated. The aq phase wasextracted with EtOAc (3×50 mL) and the combined organic phases dried(Na₂SO₄), filtered and evaporated to give nucleoside 41 as an off-whitesolid. The product was dissolved in anhyd pyridine (50 mL) and benzoylchloride (0.87 mL, 7.5 mmol) was added. The reaction was stirred for 3 hunder argon and then concentrated in vacuo. The residue was diluted withEtOAc (100 mL) and extracted with sat. aq NaHCO₃ (100 mL). The phaseswere separated and the aq phase extracted with EtOAc (2×100 ml). Thecombined organic phases were washed with brine (200 ml) and dried(Na₂SO₄). Filtration and evaporation of the organic phase produced aclear oil 42 that was dissolved in THF (100 mL). LiOH (aq, 1.0 M, 25 mL)was added and the reaction was stirred for 2 h. The reaction mixture wastransferred to a separatory funnel with EtOAc (100 mL) and brine (100mL) and extracted with EtOAc (2×100 ml). The combined organic phaseswere washed with brine (200 ml) and dried (Na₂SO₄). Filtration andevaporation in vacou gave a yellow foam that was purified by DCVC (Ø 4cm, 50-100% EtOAc, n-heptane v/v (the column was pretreated with 1% Et₃Nin heptane v/v), 5% increments, 100 mL fractions)) affording nucleoside43 (1.12 g, 65%) as a white solid. R_(f)=0.56 (EtOAc); ESI-MS m/z found689.3 ([MH]⁺, calcd. 689.3); ¹H NMR (DMSO-d₆) δ8.16 (s, 2H, Bz), 7.86(s, 1H, H6), 7.61-7.44 (m, 5H, Bz, DMT), 7.36-7.24 (m, 7H, Bz, DMT),6.92 (dd, 4H, J=9.0, 2.4, DMT), 5.64 (s, 1H, H1′), 5.41 (d, J=5.3, 1H,H3′), 4.14 (d, J=5.3, 1H, H2′), 5.64 (s, 1H, H1′), 3.75 (s, 6H, OCH₃),3.39 (d, J=10.8, 1H, H5′), 3.28 (d, J=10.8 Hz, 1H, H5′), 2.89 (d, J=9.5,1H, H1″), 2.59 (s, 3H, NCH₃), 2.58 (d, J=9.2, 1H, H1″), 1.73 (s, 3H,CH₃); ¹³C NMR (DMSO-d₆) E 178.2 (PhC(O)), 160.3 (C4), 158.2 (Ph), 147.0(C2), 144.8 (Ph), 137.4 (C6), 135.4, 135.2, 132.5, 129.9, 129.3, 128.4,128.0, 127.7, 126.9, 113.3 (Ph), 108.6 (C5), 88.9 (C1′), 85.7 (C4′),85.0 (Ph), 70.5 (C3′), 67.0 (C5′), 59.6, 58.6 (C2′, C1″), 55.1 (OCH₃),40.1 (NCH₃), 14.1 (CH₃); Anal. calcd. for C₄₀H₄₀N₄O₇: C, 69.7; H, 5.9;N, 8.1. Found: C, 69.5; H, 5.9; N, 7.7.

(1R,3R,4R,7S)-1-(4,4′-Dimethoxytrityloxymethyl)-7-(2-cyanoethoxy(diisopropylamino)phosphinoxy)-5-N-methyl-3-(4-N-benzoyl-5-methyl-cytosine-1-yl)-2-oxa-5-azabicyclo[2:2:1]heptane(44)

Compound 43 (0.50 g, 0.73 mmol) was dissolved in anhyd dichloromethane(10 mL) and 4,5-dicyanoimidazole in MeCN (1.0 M, 0.51 mL, 0.51 mmol) wasadded at ambient temperature with stirring.2-Cyanoethyl-N,N,N′N′-tetraisopropylphosphorodiamidite (0.23 mL, 0.74mmol) was added dropwise to the reaction mixture. After 2 h the reactionwas diluted with dichloromethane (20 mL) and transferred to a separatoryfunnel and extracted with sat. aq NaHCO₃ (2×30 mL) and brine (30 mL).The combined aq phases were extracted with dichloromethane (30 mL). Theorganic phases were pooled and dried (Na₂SO₄). After filtration theorganic phase was evaporated in vacuo to give a yellow foam.Purification by DCVC (Ø 4 cm, 0-100% EtOAc, n-heptane, 0.5% Et₃N v/v/v(the column was pretreated with 1% Et₃N in heptane v/v), 5% increments,50 mL fractions) afforded nucleoside 44 (0.58 g, 92%) as a white solid.R_(f)=0.67 (20% heptane, 79.5% EtOAc, 0.5% Et₃N, v/v/v); ESI-MS m/zfound 889.2 ([MH]⁺, calcd 889.4); ³¹P NMR (DMSO-d₆) δ148.4, 147.4

1-(3-O-Benzoyl-5-O-methanesulfonyl-4-C-methanesulfonyloxymethyl-β-D-threo-pentofuranosyl)thymine(52)

Anhydronucleoside 29 (30.00 g, 58.1 mmol) was heated to 70° C. in amixture of methanol (1000 ml) and acetone (1000 ml) until a clearsolution was obtained and the solution was allowed to reach roomtemperature. The reaction flask was flushed with argon and Pd/C (10 wt.% Pd on carbon, 6.2 g, 5.8 mmol) was added. The mixture was stirredvigorously under an atmosphere of hydrogen gas (balloon). After 23 h theslurry was filtered through a pad of celite. The catalyst was recoveredfrom the celite and refluxed in DMF (1000 ml) for 1 h. The hot DMFslurry was filtered through a pad of celite and the organic phasespooled and evaporated in vacuo to give2,2′-anhydro-1-(3-hydroxy-5-O-nnethanesulfonyl-4-C-methanesulfonyloxymethyl-β-D-threo-pentofuranosyl)thymine(50) as a yellow powder. Residual solvents were removed on a high vacuumpump overnight. The crude nucleoside 50 (23 g) was heated to 70° C. inDMF (300 ml) to give a clear yellow solution that was allowed to cool toroom temperature. Benzoyl chloride (81.7 g, 581 mmol, 67.4 ml) was addedfollowed by anhydrous pyridine (70 ml). After 18 h the reaction wasquenched with methanol (200 ml) and excess methanol was removed invacuo. To the dark brown solution of nucleoside 51(2,2′-anhydro-1-(3-O-benzoyl-5-O-methanesulfonyl-4-C-methanesulfonyloxymethyl-β-D-threo-pentofuranosyl)thymine)aqueous H₂SO₄ (0.25 M, 400 ml) was added. The solution was heated to 80°C. on an oil bath (At approx 50° C. precipitation occurs. The solutionbecomes clear again at 80° C.). After 22 h at 80° C. the solution wasallowed to cool down to room temperature. The reaction mixture wastransferred to a separatory funnel with EtOAc (1000 ml). The organicphase was extracted with sat. aq. NaHCO₃ (2×1000 ml). The combinedaqueous phases were extracted with EtOAc (1000+500 ml). The organicphases were pooled and extracted once more with sat. aq. NaHCO₃ (1000ml), dried (Na₂SO₄), filtered and evaporated in vacuo to give a yellowliquid. Residual solvents were removed on a high vacuum pump overnightto give a yellow syrup. The product was purified by Dry Column VacuumChromatography (Ø 10 cm, 50-100% EtOAc in n-heptane (v/v), 100 mlfractions, 10% increments, followed by 2-24% MeOH in EtOAc (v/v), 100 mlfractions, 2% increments). Fractions containing the product werecombined and evaporated in vacuo affording nucleoside 52 (25.1 g, 79%)as a white foam. R_(f)=0.54 (5% MeOH in EtOAc, v/v); ESI-MS m/z found549.0 ([MH]⁺, calcd 549.1); ¹H NMR (DMSO-d₆) δ11.39 (br s, 1H, NH),8.10-8.08 (m, 2H, Ph), 7.74-7.70 (m, 1H, Ph), 7.60-7.56 (m, 2H, Ph),7.51 (d, J=1.1, 1H, H6), 6.35 (d, J=4.9, 1H, H1′), 6.32 (d, J=5.3, 1H,2′-OH), 5.61 (d, J=4.0, 1H, H3′), 4.69 (d, J=10.8, 1H), 4.59 (m, 1H,H2′), 4.55 (d, J=10.8, 1H), 4.52 (d, J=10.8, 1H), 4.46 (d, J=10.6, 1H)(H5′ and H1″), 3.28 (s, 3H, Ms), 3.23 (s, 3H, Ms), 1.81 (s, 3H, CH₃);¹³C NMR (DMSO-d₆) δ164.5, 163.6 (C4, PhC(O)), 150.3 (C2), 137.7 (C6),133.8, 129.6, 128.7, 128.6 (Ph), 108.1 (C5), 84.8 (C1′), 81.1 (C4′),78.0 (C3′), 73.2 (C2′), 68.0, 67.1 (C5′, C1″), 36.7, 36.6 (Ms), 11.9(CH₃); Anal. calcd for C₂₀H₂₄N₂O₁₂S₂.0.33 H₂O: C, 44.34; H, 4.65; N,4.85. Found: C, 44.32; H, 4.58; N, 4.77.

(1R,3R,4R,7R)-7-Benzoyloxy-1-methansulfonyloxymethyl-3-(thymin-1-yl)-2-oxa-5-thiabicyclo[2:2:1]heptane(54)

1-(3-O-Benzoyl-5-O-methanesulfonyl-4-C-methanesulfonyl-oxymethyl-β-D-threo-pentofuranosyl)thymine(52) (10.00 g, 18.23 mmol) was dissolved in anhydrous dichloromethane(500 ml) and cooled to 0° C. Pyridine (15 ml) and DMAP (8.91 g, 72.9mmol) was added followed by dropwise addition oftrifluoromethanesulfonic anhydride (10.30 g, 36.5 mmol, 6.0 ml). After 1h the reaction was quenched with sat. aq. NaHCO₃ (500 ml) andtransferred to a separatory funnel. The organic phase was extracted with1.0 M aq HCl (500 ml), sat. aq NaHCO₃ (500 ml) and brine (500 ml). Theorganic phase was evaporated in vacuo with toluene (100 ml) to give1-(3-O-benzoyl-5-O-methanesulfonyl-4-C-methanesulfonyloxymethyl-2-O-trifluoromethanesulfonyl-β-D-threo-pentofuranosyl)thymine(53) as a yellow powder. The crude nucleoside 53 was dissolved inanhydrous DMF (250 ml) and Na₂S (1.57 g, 20.1 mmol) was added to give adark green slurry. After 3 h the reaction was quenched with half sat.aq. NaHCO₃ (500 ml) and extracted with CH₂Cl₂ (500+2×250 ml). Thecombined organic phases were extracted with brine (500 ml), dried(Na₂SO₄), filtered and concentrated in vacuo to give a yellow liquid.Residual solvent was removed overnight on a high vacuum pump to give ayellow gum that was purified by Dry Column Vacuum Chromatography (Ø 6cm, 50-100% EtOAc in n-heptane (v/v), 50 ml fractions, 10% increments,followed by 2-20% MeOH in EtOAc (v/v), 50 ml fractions, 2% increments)to give nucleoside 54 (6.15 g, 72%) as a yellow foam.

R_(f)=0.27 (20% n-heptane in EtOAc, v/v); ESI-MS m/z found 469.0 ([MH]⁺,calcd 469.1); ¹H NMR (CDCl₃) δ 8.70 (br s, 1H, NH), 8.01-7.99 (m, 2H,Ph), 7.67 (d, J=1.1, 1H, H6), 7.65-7.61 (m, 1H, Ph), 7.50-7.46 (m, 2H,Ph), 5.98 (s, 1H, H1′), 5.34 (d, J=2.4, 1H, H3′), 4.66 (d, J=11.7, 1H,H5′ a), 4.53 (d, J=11.5, 1H, H5′ b), 4.12 (m (overlapping with residualEtOAc), 1H, H2′), 3.15-3.13 (m, 4H, H1″a and Ms), 3.06 (d, J=10.6, 1H,H1″b), 1.98 (d, J=1.1, 3H, CH₃); ¹³C NMR (CDCl₃) δ 165.2, 163.5 (C4,PhC(O)), 149.9 (C2), 134.1, 133.9, 129.8, 128.7, 128.3 (C6, Ph), 110.7(C5), 91.1 (C1′), 86.8 (C4′), 72.6 (C3′), 65.8 (C5′), 50.5 (C2′), 37.9(Ms), 35.1 (C1″), 12.5 (CH₃); Anal. calcd for C₁₉H₂₀N₂O₈S₂.0.33 EtOAc:C, 49.21; H, 4.72; N, 5.47. Found: C, 49.25; H, 4.64; N, 5.48.

(1R,3R,4R,7R)-7-Benzoyloxy-1-benzoyloxymethyl-3-(thymin-1-yl)-2-oxa-5-thiabicyclo[2:2:1]heptane(55)

Nucleoside 54 (1.92 g, 4.1 mmol) was dissolved in anhydrous DMF (110ml). Sodium benzoate (1.2 g, 8.2 mmol) was added and the mixture washeated to 100° C. for 24 h. The reaction mixture was transferred to aseparatory funnel with half sat. brine (200 ml) and extracted with EtOAc(3×100 ml). The combined organic phases were dried (Na₂SO₄), filteredand evaporated in vacuo to give a brown liquid. The product was put on ahigh vacuum pump to remove residual solvent. The resulting brown gum waspurified by Dry Column Vacuum Chromatography (Ø 4 cm, 0-100% EtOAc inn-heptane (v/v), 50 ml fractions, 10% increments, followed by 2-10% MeOHin EtOAc (v/v), 50 ml fractions, 2% increments) to afford nucleoside 55(1.64 g, 81%) as a slightly yellow foam. R_(f)=0.57 (20% n-heptane inEtOAc, v/v); ESI-MS m/z found 495.1 ([MH]⁺, calcd 495.1); ¹H NMR (CDCl₃)δ 9.02 (br s, 1H, NH), 8.07-7.99 (m, 4H, Ph), 7.62-7.58 (m, 2H, Ph),7.47-7.42 (m, 5H, Ph and H6), 5.95 (s, 1H, H1′), 5.46 (d, J=2.2, 1H,H3′), 4.93 (d, J=12.8, 1H, H5′ a), 4.60 (d, J=12.8, 1H, H5′ b), 4.17 (d,J=2.2, 1H, H2′), 3.27 (d, J=10.6, 1H, H1″a), 3.16 (d, J=10.6, 1H, H1″b),1.55 (d, J=1.1, 3H, CH₃); ¹³C NMR (CDCl₃) δ 165.8, 165.1, 163.7 (C4,2×PhC(O)), 150.0 (C2), 133.9, 133.7, 133.6, 129.8, 129.6, 129.0, 128.8,128.6, 128.5 (C6, 2×Ph), 110.3 (C5), 91.3 (C1′), 87.5 (C4′), 72.9 (C3′),61.3 (C5′), 50.6 (C2′), 35.6 (C1″), 12.3 (CH₃); Anal. calcd forC₂₅H₂₂N₂O₇S: C, 60.72; H, 4.48; N, 5.66. Found: C, 60.34; H, 4.49; N,5.35.

(1R,3R,4R,7R)-7-Hydroxy-1-hydroxymethyl-3-(thymin-1-yl)-2-oxa-5-thiabicyclo[2:2:1]heptane(56)

Nucleoside 55 (1.50 g, 3.0 mmol) was dissolved in methanol saturatedwith ammonia (50 ml). The reaction flask was sealed and stirred atambient temperature for 20 h. The reaction mixture was concentrated invacuo to give a yellow gum that was purified by Dry Column VacuumChromatography (Ø 4 cm, 0-16% MeOH in EtOAc (v/v), 1% increments, 50 mlfractions) affording nucleoside 56 (0.65 g, 76%) as clear crystals.R_(f)=0.31 (10% MeOH in EtOAc, v/v); ESI-MS m/z found 287.1 ([MH]⁺,calcd 287.1); ¹H NMR (DMSO-d₆) δ11.32 (br s, 1H, NH), 7.96 (d, J=1.1,1H, H6), 5.95 (s, 1H, H6), 5.70 (d, J=4.2, 1H, 3′-OH), 5.62 (s, 1H,H1′), 4.49 (t, J=5.3, 1H, 5′-OH), 4.20 (dd, J=4.1 and 2.1, 1H, H3′),3.77-3.67 (m, 2H, H5′), 3.42 (d, J=2.0, 1H, H2′), 2.83 (d, J=10.1, 1H,H1″a), 2.64 (d, J=10.1, 1H, H1″b), 1.75 (d, J=1.1, 3H, CH₃); ¹³C NMR(DMSO-d₆) δ 163.8 (C4), 150.0 (C2), 135.3 (C6), 107.5 (C5), 90.2, 89.6(C1′ and C4′), 69.4 (C3′), 58.0 (C5′), 52.1 (C2′), 34.6 (C1″), 12.4(CH₃); Anal. calcd for C₁₁H₁₄N₂O₆S: C, 46.15; H, 4.93; N, 9.78. Found:C, 46.35; H, 4.91; N, 9.54.

(1R,3R,4R,7R)-1-(4,4′-Dimethoxytrityloxymethyl)-7-hydroxy-5-methyl-3-(thymin-1-yl)-2-oxa-5-thiabicyclo[2:2:1]heptane(57)

Nucleoside 56 (0.60 g, 2.1 mmol) was dissolved in anhydrous pyridine (10ml). 4,4′-Dimethoxytrityl chloride (0.88 g, 2.6 mmol) was added and thereaction was stirred at ambient temperature for 3 h. The reactionmixture was transferred to a separatory funnel with water (100 ml) andextracted with EtOAc (100+2×50 ml). The combined organic phases werewashed with sat. aq NaHCO₃ (100 ml), brine (100 ml) and evaporated todryness in vacuo to give a viscous yellow liquid. The product wasredissolved in toluene (50 ml) and concentrated in vacuo to give ayellow foam. The foam was dried on a high vacuum pump overnight andpurified by Dry Column Vacuum Chromatography (Ø 4 cm, 10-100% EtOAc inn-heptane (v/v), 10% increments, 50 mL fractions) affording nucleoside57 (1.08 g, 88%) as a white foam. R_(f)=0.24 (20% n-heptane in EtOAc,v/v); ESI-MS m/z found 587.1 ([M−H]⁺, calcd 587.19); ¹H NMR (CDCl₃) δ8.96 (br s, 1H, NH), 7.74 (d, J=1.1, 1H, H6), 7.46-7.44 (m, 2H, Ph),7.35-7.22 (m, 9H, Ph), 7.19-7.7.15 (m, 2H, Ph), 6.86-6.80 (m, 2H, Ph),5.82 (s, 1H, H1′), 4.55 (dd, J=9.3 and 2.1, 1H, H3′), 3.79 (s, 6H,OCH₃), 3.71 (d, J=2.0, 1H, H2′), 3.50 (s, 2H, H5′), 2.81 (d, J=10.8, 1H,H1″a), 2.77 (d, J=10.8, 1H, H1″b), 2.69 (d, J=9.2, 1H, 3′-OH), 1.42 (s,3H, CH₃); ¹³C NMR (CDCl₃) δ 158.7 (C4), 150.1 (C2), 144.1, 135.2, 135.1,130.1, 129.1, 128.1, 128.0, 127.1, 127.0 (C6, Ph), 113.3 (Ph), 110.0(C5), 90.2 (C(Ph)₃), 89.6 (C1′), 87.0 (C4′), 71.7 (C3′), 60.9 (C5′),55.2 (C2′), 34.7 (C1″), 12.2 (CH₃); Anal. calcd for C₃₂H₃₂N₂O₇S.0.5 H₂O:C, 64.31; H, 5.57; N, 4.69. Found: C, 64.22; H, 5.67; N, 4.47.

(1R,3R,4R,7R)-7-(2-Cyanoethoxy(diisopropylamino)phosphinoxy)-1-(4,4′-dimethoxytrityloxymethyl)-3-(thymin-1-yl)-2-oxa-5-thiabicyclo[2.2.1]heptane(58)

Nucleoside 57 (0.78 g, 1.33 mmol) was dissolved in anhydrousdichloromethane (5 ml) and a 1.0 M solution of 4,5-dicyanoimidazole inacetonitrile (0.93 ml, 0.93 mmol) was added followed by dropwiseaddition of 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite(0.44 ml, 1.33 mmol). After 2 h the reaction was transferred to aseparatory funnel with dichloromethane (40 ml) and extracted with sat.aq NaHCO₃ (2×25 ml) and brine (25 ml). The organic phase was dried(Na₂SO₄), filtered and evaporated in vacuo to give nucleoside 58 (1.04g, 99%) as a white foam.

R_(f)=0.29 and 0.37—two diastereoisomers (20% n-heptane in EtOAc, v/v);ESI-MS m/z found 789.3 ([MH]⁺, calcd 789.30); ³¹P NMR (DMSO-d₆) δ150.39, 150.26

(1R,3R,4R,7S)-7-Benzyloxy-1-methansulfonyloxymethyl-3-(thymin-1-yl)-2-oxa-5-thiabicyclo[2:2:1]heptane(60).

Nucleoside 31 (0.10 g, 0.17 mmol) was dissolved in anhyd DMF (1 mL) andpotassium thioacetate (25 mg, 0.22 mmol) was added. The reaction wasstirred at ambient temperature for 5 h and transferred to a separatoryfunnel with brine (10 mL). The aq phase was extracted withdichloromethane (3×10 mL) and the combined organic phases dried(Na₂SO₄), filtered and evaporated in vacuo to give a yellow liquid. Thecrude product 59 was dissolved in THF (2 mL) and LiOH.H₂O (35 mg in 1 mLwater, 0.84 mmol) was added. After 20 min the reaction was completed andquenched by the addition of glacial acetic acid (0.5 mL). The THF wasremoved in vacuo and the residue dissolved in dichloromethane (10 mL)and extracted with sat. aq NaHCO₃ (2×10 mL). The aq phases wereextracted with dichloromethane (10 mL). The combined organic phases weredried (Na₂SO₄), filtered and evaporated in vacuo to give a yellow liquidthat was purified by DCVC (Ø 1 cm, 0-80% EtOAc in n-heptane v/v, 2.5%increments, 10 mL fractions). Fractions containing nucleoside 60 werecombined and evaporated in vacuo to afford a white powder (36 mg, 47%from 31). R_(f)=0.38 (80% EtOAc in n-heptane, v/v); ESI-MS m/z found455.0 ([MH]⁺, calcd 455.1); ¹H NMR (DMSO-d₆) δ11.38 (br s, 1H, NH), 7.50(d, J=1.1, 1H, H6), 7.36-7.27 (m, 5H, Ph), 5.77 (s, 1H, H1′), 4.68 (d,J=11.7, 1H), 4.61 (d, J=11.7, 1H), 4.60 (d, J=11.7, 1H), 4.56 (d,J=11.5, 1H) (H5′, CH ₂Ph), 4.20 (d, J=1.8, 1H, H3′), 4.00 (d, J=2.0, 1H,H2′), 3.29 (s, 3H, Ms), 3.02 (d, J=10.6, 1H, H1″a), 2.90 (d, J=10.4, 1H,H1″b), 1.78 (s, 3H, CH₃); ¹³C NMR (DMSO-d₆) δ 163.9 (C4), 150.1 (C2),137.5, 134.1, 128.3, 127.7 (C6, Ph), 108.3 (C5), 90.5 (C1′), 86.6 (C4′),76.9 (C3′), 70.9, 66.8 (C5′, CH₂Ph), 49.5 (C2′), 36.8 (Ms), 35.1 (C1′),12.3 (CH₃); Anal. calcd for C₁₉H₂₂N₂O₇₅₂.0.33EtOAc: C, 50.5; H, 5.1; N,5.8. Found: C, 50.8; H, 5.1; N, 5.8.

9-(3-O-benzyl-5-O-(methanesulfonyl)-4-C-[[(methanesulfonyl)oxy]methyl]-2-O-trifluormethanesulfonyl-α-L-threo-pentofuranosyl)-6-N-benzoyladenine(62)

Compound 61¹ (9.58 g, 15 mmol) was concentrated from dry acetonitrile inorder to remove residual water. The residue was dissolved in drydichloromethane (100 ml) and cooled to −30° C. while stirred under Ar.The solution was added dry pyridine (3.6 ml, 44 mmol), followed bydropwise addition of Tf₂O (3.7 ml, 22 mmol). The reaction mixture wasallowed to reach 0° C. TLC (eluent: EtOAc) shows full conversion toproduct (R_(f)=0.66). The reaction was quenched by addition of sat.NaHCO₃-soln. (100 ml) and diluted with dichloromethane (100 ml). Thelayers were separated and the org. layer was washed with sat.NaHCO₃-soln (100 ml), brine (100 ml), dried (Na₂SO₄), filtered and thesolvent removed in vacuo to afford an orange foam, which was purified bydry column chromatography (eluent: Heptane→EtOAc) to afford puretriflate 62 (8.53 g, 74% yield). R_(f)=0.60 (eluent: EtOAc). ESI-MS m/zfound 780.0 ([MH]⁺, calcd 780.0); ¹H-NMR (CDCl₃, 400 MHz): δ 9.05 (1H,s, N—H), 8.80 (1H, s, base), 8.21 (1H, s, base), 8.00 (2H, d, J=7.3 Hz,Bz), 7.61 (1H, t, J=7.3 Hz, Bz), 7.52 (2H, t, J=7.3 Hz, Bz), 7.41-7.30(5H, m, Bn), 6.56 (1H, t, J=5.5 Hz, H-2′), 6.34 (1H, d, J=5.5 Hz, H-1′),4.81 (2H, d, J=10.4 Hz, CH₂), 4.73 (1H, d, J=5.9 Hz, H-3′), 4.65 (1H, d,J=11.3 Hz, CH₂), 4.44 (1H, d, J=11.3 Hz, CH₂), 4.34 (1H, d, J=11.1 Hz,CH₂), 4.14 (1H, d, J=11.4 Hz, CH₂), 3.05 (3H, s, OMs), 2.91 (3H, s,OMs); ¹³C-NMR (CDCl₃, 100 MHz): δ 164.34, 152.94, 151.26, 149.88,141.55, 135.07, 133.24, 132.84, 128.98, 128.83, 128.80, 128.49, 127.70,86.49, 85.03, 83.62, 80.33, 74.49, 67.51, 67.22, 37.76 (OMs), 37.41(OMs);¹ Compound 61 was made according to procedure described in JACS,124, p. 2164-2176 (2002). Triflate 62 is also described in this article,but not as an isolated product.

(1S,3R,4S,7R)-7-benzyloxy-3-(6-N-benzoyladenin-9-yl)-2,5-dioxabicyclo[2.2.1]heptane(53)

Pure 62 (100 mg, 0.128 mmol) was dissolved in THF (7 ml), cooled to 0°C. and added 1 M LiOH (1.3 ml, 10 equiv.). The reaction mixture wasallowed to slowly reach r.t. When LCMS confirmed full conversion of 62to 63, the reaction was neutralized with 1 M HCl satd. with NaCl (1.3ml), diluted with DCM (20 ml) and brine (10 ml). Layers were separatedand the aqueous layer was extracted with DCM (2×20 ml). Comb. organiclayers were dried (Na₂SO₄), filtered and the solvent removed in vacuo toafford a clear oil (63)² (quantitative yield). R_(f)=0.49 (Eluent:EtOAc). ESI-MS m/z found 552.2 ([M1-1]⁺, calcd 552.1); ¹H-NMR (CDCl₃,400 MHz): 8.64 (1H, s, N—H), 8.44 (1H, s, Adenin), 7.95 (2H, d, J=7.1Hz, Bz), 7.50 (1H, t, J=7.3 Hz, Bz), 7.40 (1H, t, J=7.3 Hz, Bz),7.07-6.79 (5H, m, OBn), 6.11 (1H, s, H-1′), 4.66 (1H, d, J=11.5 Hz,CH₂), 4.61 (1H, d, J=11.5 Hz, CH₂), 4.48 (1H, d, J=1.8 Hz, H-2′/H-3′),4.30 (1H, d, J=11.9 Hz, CH₂), 4.12 (1H, d, J=11.9 Hz, CH₂), 4.07 (1H, d,J=1.8 Hz, H-3′/H-2′), 4.02 (1H, d, J=8.6 Hz, CH₂), 3.94 (1H, d, J=8.6Hz, CH₂), 3.02 (3H, s, OMs); ¹³C-NMR (CDCl₃, 100 MHz): δ 165.31, 152.03,150.45, 148.54, 141.99, 135.38, 132.90, 132.84, 128.63, 128.37, 128.26,127.98, 127.88, 121.34, 87.90, 86.16, 79.84, 76.29, 73.45, 72.51, 67.76,64.47, 37.48 (OMs). ²Compound 63 is also described in JACS 124, p.2164-2176 (2002) but not as an isolated product.

1-(2-azido-3-O-benzyl-4-C-methanesulfonyloxymethyl-5-O-methanesulfonyl-2-deoxy-α-L-erythro-pentofuranosyl)-6-benzoyladenine-9-yl (64)

Not quite pure 62 (6.23 g, 0.008 mol) was dissolved in dry DMF (70 ml),added NaN₃ (5.2 g, 10 equiv.) and allowed to stir at r.t. for 3 days.Quenched by addition of water (100 ml) and diluted with DCM (200 ml).Layers were separated and the org. layer was washed with brine (2×125ml), dried (Na₂SO₄) and the solvent removed in vacuo. The residue waspurified by dry column liquid chromatography (eluent: heptane→EtOAc) toafford pure 64 (5.38 g, quantitative yield). R_(f)=0.60 (Eluent: EtOAc).ESI-MS m/z found 673.0 ([MH]⁺, calcd 673.1); ¹H-NMR (CDCl₃, 400 MHz): δ9.14 (1H, s), 8.70 (1H, s), 8.93 (1H, s), 8.00 (3H, d, J=7.3 Hz),7.59-7.50 (3H, 2×t, J=7.3 Hz), 7.41-7.37 (5H, m), 6.51 (1H, d, J=4 Hz,H-1), 4.92 (1H, d, J=11.7 Hz), 4.77 (1H, d, J=11.3 Hz), 4.75 (1H, d,J=4.8 Hz, H-3), 4.70 (1H, d, J=11.3 Hz), 4.50 (1H, dd, J=4.2 Hz, J=4.6Hz, H-2), 4.41 (2H, d, J=11-12 Hz), 4.27 (1H, d, J=11 Hz), 3.05 (3H, s,OMs), 3.02 (3H, s, OMs). ¹³C-NMR (CDCl₃, 100 MHz): δ 164.4, 162.3,152.5, 151.1, 149.3, 142.1, 135.5, 133.3, 132.6, 128.9, 128.8, 128.8,128.7, 128.4, 127.6, 122.3 (A^(Bz) and OBn), 82.35, 81.79, 79.55, 74.58(OBn), 68.51, 68.06, 62.59, 37.78 (OMs), 37.57 (OMs)

(1S,3R,4R,7S)-7-Benzyloxy-1-methansulfonyloxymethyl-3-(6-benzoyladenin-9-yl)-2-oxa-5-azabicyclo[2:2:1]heptane(65).

To a solution of 64 (2.28 g, 3.4 mmol) in THF (100 ml) at rt aq NaOH(2.0 M, 34 ml) and PMe₃ in THF (1.0 M, 7 ml) was added with stirring.After over night at r.t. the THF was partly removed under reducedpressure. Brine (100 mL) and EtOAc (200 mL) was added and the phaseswere separated. The org. layer was washed with brine (100 ml). The comb.aqueous layer was extracted with dichloromethane (200 mL). The combinedorganic phases were dried (Na₂SO₄), filtered and concentrated in vacuoto give a yellow foam (1.73 g) which was purified by dry column liquidchromatography to afford pure nucleoside 65 (848 mg) as a yellow foam.R_(f)=0.13 (EtOAc). *Comb. with residues from similar reactions beforepurification; R_(f)=0.21 (eluent: EtOAc). ESI-MS m/z found 551.1 ([MH]⁺,calcd 551.1); ¹H NMR (DMSO-d₆, 400 MHz): δ 11.18 (1H, br s, NH), 8.77(1H, s, A^(Bz)), 8.73 (1H, s, A^(Bz)), 8.06 (2H, d, J=7.3 Hz), 7.64 (1H,t, J=7.3 Hz, Bz), 7.55 (2H, t, J=7.3 Hz, Bz), 7.45 (2H, d, J=7.2 Hz,Bn), 7.38 (2H, t, J=7.2 Hz, Bn), 7.31 (1H, t, J=7.2 Hz, Bn), 6.52 (1H,d, J=1.6 Hz, H-1′), 4.74 (1H, d, J=11.9 Hz, H-5′ a/H-1″a), 4.65 (1H, d,J=11.9 Hz, H-5′ b/H-1″b), 4.59 (1H, d, J=11.9 Hz, H-1″a/H-5′ a), 4.52(1H, d, J=11.8 Hz, H-1″b/H-5′ b), 4.44 (1H, s, H-3′), 4.04 (1H, d, J=7.2Hz, Bn), 4.01 (1H, d, J=7.2 Hz, Bn), 3.91 (1H, br s, H-2′), 3.22 (3H, s,OMs); ¹³C-NMR (DMSO-d₆, 100 MHz): δ 170.34, 165.59, 152.12, 151.47,150.09, 143.20, 137.89, 133.44, 132.43, 128.48, 128.31, 127.70, 125.19(Bz and Bn), 87.30, 84.45, 80.47, 71.13 (Bn), 66.99, 59.92, 59.80,51.27, 36.93 (OMs)

2′,3′-epoxide (66)

To a solution of 62 (50 mg) in dry DCM (1.5 ml) at r.t. was added MsOH(0.5 ml) dropwise. Reaction was stirred at r.t. until full conversion ofs.m. was confirmed by LCMS. Reaction was diluted with DCM (20 ml),cooled to 0° C., neutralized with Et₃N (1.1 ml), washed with sat.NaHCO₃-soln (20 ml), brine (20 ml), dried (Na₂SO₄), filtered and thesolvent removed in vacuo to afford a clear oil (66) (49 mg, quantitativeyield). R_(f)=0.24 (eluent: EtOAc). ESI-MS m/z found 540.2 ([MH]⁺, calcd540.1); ¹H NMR (CDCl₃, 400 MHz): δ 9.3 (1H, br s, N—H), 8.67 (1H, s,base), 8.33 (1H, s, base), 7.94 (2H, d, J=7.5 Hz), 7.51 (1H, t, J=7.4Hz), 7.42 (2H, t, J=7.5 Hz), 6.61 (1H, s, H-1′), 4.57 (1H, d, J=11.3Hz), 4.47 (1H, d, J=10.8 Hz), 4.44 (1H, d, J=11.3 Hz), 4.36 (1H, d,J=10.8 Hz), 4.25 (1H, d, J=2.7 Hz, H-2′/H-3′), 4.13 (1H, d, J=2.7 Hz,H-3′/H-2′), 3.11 (3H, s, OMs), 3.01 (3H, s, OMs); ¹³C-NMR (CDCl₃, 100MHz): δ 164.7, 152.6, 151.5, 149.4, 141.3, 133.2, 132.6, 128.6, 128.6,128.3, 128.3, 127.7, 122.2 (A^(Bz)), 81.45, 81.23, 68.64, 66.58, 57.59,57.27, 37.66 (OMs), 37.50 (OMs);

1-(2-azido-3-O-benzyl-4-C-methanesulfonyloxymethyl-5-O-methanesulfonyl-2-deoxy-α-L-threo-pentofuranosyl)-6-benzoyladenine-9-yl (67)

To a solution of 66 (50 mg, 0.093 mmol) in anh. DMF (2 ml) was addedNaN₃ (60 mg, 10 equiv.). The mixture was heated to 50° C. overnight.LCMS confirms full conversion of 66 to 67. Reaction mixture was dilutedwith water (15 ml) and DCM (15 ml). Layers were separated and the org.layer was washed with brine (15 ml), dried (Na₂SO₄), filtered and thesolvent removed in vacuo to afford 67 (43 mg, 80% yield). R_(f)=0.51(eluent: EtOAc). ESI-MS m/z found 583.0 ([MH]⁺, calcd 583.1); ¹H NMR(CDCl₃, 400 MHz): δ 11.27 (1H, s, N—H), 8.79 (1H, s, base), 8.05 (2H, d,J=7.3 Hz, Bz), 7.95 (1H, s, base), 7.65 (1H, t, J=7.5 Hz), 7.55 (2H, t,J=7.5 Hz), 6.70 (1H, d, J=5.5 Hz, H-1′), 6.18 (1H, d, J=8.6 Hz, 3′-OH),5.27 (1H, t, J=8.6 Hz, H-3′), 4.66 (1H, d, J=10.7 Hz, CH₂), 4.57 (1H,dd, J=5.6 Hz, J=8.5 Hz, H-2′), 4.47 (1H, d, J=10.8 Hz, CH₂), 4.41 (2H,d, J=10.8 Hz, CH₂), 3.29 (3H, s, OMs), 3.22 (3H, s, OMs); ¹³C-NMR(CDCl₃, 100 MHz): δ 165.67, 162.33, 152.33, 152.20, 150.75, 142.63,133.28, 132.56, 128.57, 128.52, 125.52, 82.34, 81.79, 74.77, 69.00,68.46, 66.11, 36.87 (OMs), 35.85 (OMs)

(1R,3R,4R,7S)-7-hydroxy-1-methansulfonyloxymethyl-3-(6-benzoyladenin-9-yl)-2-oxa-5-thiobicyclo[2:2:1]heptane(68)

Compound 66 (1.0 g, 1.9 mmol) was dissolved in dry DMF and added Na₂S(290 mg, 2 equiv.). Reaction turns green. Allowed to stir at r.t.overnight. LCMS confirms full conversion of compound 1. Reaction mixturewas partitioned between brine (100 ml) and EtOAc (100 ml). Layers wereseparated and the aq. layer was extracted with EtOAc (2×100 ml) and DCM(2×100 ml). Combined organic layer was washed with brine (200 ml), dried(Na₂SO₄), filtered and the solvent removed in vacuo. Residue waspurified by DCLC to afford compound 2 (268 mg, 30% yield). LCMS: found478.0, calc. 478.0 (M+H). ¹H-NMR (CDCl₃, 400 MHz): δ 9.5-7.3 (8H,A^(Bz)), 6.46 (1H, s, H-1), 4.64 (2H, 2×d, J=11.4 Hz, H-1″a and b), 4.56(1H, d, J=1 Hz, H-3′), 3.75 (1H, d, J=1 Hz, H-2′), 3.04, 5.97 (2H, 2×d,J=10.8 Hz, H-5′ a and b), 3.02 (3H, s, OMs).

(1S,3R,4S,7S)-7-Benzyloxy-1-methansulfonyloxymethyl-3-(6-benzoyladenin-9-yl)-2-oxa-5-thiobicyclo[2:2:1]heptane(69)

Compound 62 (3.30 g, 4.2 mmol) was dissolved in dry DMF (33 ml) andadded Na₂S (1.65 g, 5 equiv.). Reaction colour goes from green to orangein 30 min. LCMS confirms full conversion to compound 2. Reaction mixturewas partitioned between sat. NaHCO₃-soln. (150 ml) and DCM (150 ml).Layers were separated and the aqueous layer was extracted with DCM (2×75ml). Combined org. layer was washed with sat. NaHCO₃-soln. (150 ml),brine (150 ml), dried (Na₂SO₄), filtered and the solvent removed invacuo to afford an oily residue (˜3 g) which was used in the next stepwithout further purification. LCMS: found 568.0, calc. 568.1 (M+H).¹H-NMR (400 MHz, CDCl₃): δ 9.5 (1H, br s, N—H), 8.65 (1H, s, A^(Bz)),8.36 (1H, s, A^(Bz)), 7.99 (2H, 2×d, J=7.3 Hz, A^(Bz)), 7.54 (1H, t,J=7.3 Hz, A^(Bz)), 7.45 (2H, t, J=7.3 Hz, A^(Bz)), 7.30-7.36 (5H, m,OBn), 6.61 (1H, d, J=2.2 Hz, H-1′), 4.72 (1H, d, J=11.6 Hz, H-1″a), 4.59(1H, d, J=11.3 Hz, H-1″b), 4.59 (1H, d, J=1.6 Hz, H-3′), 4.91, (2H, s,OBn), 4.05 (1H, t, J=2.0 Hz, H-2′), 3.17 (1H, d, J=10.5 Hz, H-5′ a),3.05 (1H, d, J=11.0 Hz, H-5′ b), 3.02 (3H, s, OMs). ¹³C-NMR (100 MHz,CDCl₃): δ 152.1, 150.8, 149.2, 141.3, 136.1, 133.4, 132.5, 128.6, 128.6,128.5, 128.2, 127.8, 127.7, 123.0, (A^(Bz), OBn), 87.34 (C-4′), 87.25(C-1′), 80.35 (C-3′), 72.05 (C-1″), 66.48 (OBn), 51.80 (C-2′), 37.67(OMs), 36.00 (C-5′).

Compound 70

Compound 69 (2.38 g, 4.2 mmol) was dissolved in dry DMSO (25 ml), addedNaOBz (1.24 g, 2 equiv.) and heated to 100° C. overnight. LCMS confirmsfull conversion to compound 4. Reaction mixture was partitioned betweenwater (150 ml) and DCM (150 ml). Layers were separated and the aqueouslayer was extracted with DCM (2×100 ml). Comb. organic layer was washedwith brine (2×150 ml), dried (Na₂SO₄), filtered and concentrated ontosilica. Purified by DCLC to afford compound 3 (945 mg, 38% over twosteps). LCMS: found 594.2, calc. 594.1 (M+H). ¹H-NMR (400 MHz, CDCl₃): δ8.63-7.18 (17H, A^(Bz), OBz, OBn), 6.56 (1H, d, J=2.2 Hz, H-1′), 4.72(1H, d, J=11.5 Hz, H-1″a), 4.69 (1H, d, J=11.0 Hz, H-1″b), 4.57 (1H, d,J=1.6 Hz, H-3′), 4.53 (1H, d, J=11.6 Hz, OBn), 4.49 (1H, d, J=12 Hz,OBn), 4.01 (1H, br s, H-2′), 3.24 (1H, d, J=10.4 Hz, H-5′ a), 2.99 (1H,d, J=10.4 Hz, H-5′ b). ¹³C-NMR (100 MHz, CDCl₃): δ 165.5, 152.1, 150.7,149.2, 141.4, 136.2, 133.5, 133.2, 132.5, 129.5, 129.1, 128.6, 128.4,128.3, 128.1, 127.7, 127.6 (A^(Bz), OBz, OBn), 87.73 (C-4′), 87.32(C-1′), 80.47 (C-3′), 71.88 (C-1″), 61.73 (OBn), 51.80 (C-2′), 36.43(C-5′).

Compound 71

Compound 70 (966 mg, 1.627 mmol) was dissolved in dry DCM (27 ml) andadded MsOH (9 ml). Stirred at r.t. for 1 hr. LCMS confirms fulldebenzylation.* Reaction mixture was diluted with DCM (30 ml), washedwith brine (50 ml), sat. NaHCO₃-soln. (50 ml), dried (Na₂SO₄), filteredand the solvent removed in vacuo to afford compound 6 (739 mg, 90%yield). LCMS: found 504.1, calc. 504.1 (M+H). *Depurination is alsodetected, so cooling might be advantageous.

Compound 73

Compound 71 (739 mg, 1.468 mmol) was dissolved in THF (60 ml) and added1 M LiOH (7.5 ml). The reaction was stirred at r.t. More 1 M LiOH (1 ml)was added after 90 min. Completion of reaction was confirmed by TLC(eluent: EtOAc/MeOH 9:1) after another 60 min. The reaction was quenchedwith 1 M HCl satd. with NaCl (8.5 ml) and the mixture was partitionedbetween brine (100 ml) and EtOAc (100 ml). Layers were separated and theaqueous layer was extracted with EtOAc (2×100 ml). Combined org. layerwas washed with brine (100 ml), dried (Na₂SO₄), filtered and the solventremoved in vacuo to afford a yellow solid (compound 72), which wasco-evaporated with dry pyridine. The residue was dissolved in drypyridine (25 ml), added DMAP (180 mg, 1 equiv.) followed by DMTCI (597mg, 1.2 equiv.) and stirred at r.t. Added more DMTCI (200 mg). TLC(eluent: EtOAc/MeOH 9:1) after 24 hrs shows full conversion to compound73. Reaction was diluted with DCM (100 ml) and washed with water (100ml). Aqueous layer was extracted with DCM (50 ml) and combined org.layer was washed with sat. NaHCO₃-soln. (100 ml), brine (100 ml), dried(Na₂SO₄), filtered and the solvent removed in vacuo to afford a residuewhich was purified by DCLC to afford compound 6 (518 mg, 50% over twosteps). LCMS: found 702.2, calc. 702.2 (M+H).

Compound 74

Compound 73 (518 mg, 0.738 mmol) was dissolved in DCM (10 ml), added 1 MDCI (520 μl, 0.7 equiv., dissolved in acetonitrile) followed bybisamidite reagent (244 μl, 1 equiv.) and stirred at r.t., under anatmosphere of N₂. More bisamidite reagent was added (2×40 μl) and theflask was transferred to the fridge over weekend. LCMS confirms fullconversion to amidite. The reaction mixture was diluted with DCM (100ml), washed with sat. NaHCO₃-soln. (2×100 ml), brine (100 ml), dried(Na₂SO₄), filtered and the solvent removed in vacuo to afford compound74 (642 mg, 97% yield). LCMS: found 902.2, calc. 903.3 (M+H).

9-(2-O-3-Acetyl-3-O-benzyl-5-O-methanesulfonyl-4-C-methanesulfonyloxymethyl-β-L-threo-furanosyl)-2-amino-6-chlorpurine(75)

1,2-Di-O-acetyl-5-O-methanesulfonyl-4-C-methanesulfonyloxymethyl-3-O-benzyl-L-threo-pentofuranose(20.6 g, 40.0 mmol) is dissolved in anh. 1,2-dichloroethane (250 mL) and2-amino-6-chloropurine (7.5 g, 44.4 mmol) was added followed byN,O-bis(trimethylsilyl)acetamide (19.6 mL, 80.0 mmol). The reactionmixture was refluxed until it turned clear (1 h) and cooled to roomtemperature. Trimethylsilyl triflate (14.5 mL, 80.0 mmol) was added over15 min and the reaction mixture was refluxed for 3 h. The reactionmixture was allowed to cool to room temperature and was poured into sat.aq NaHCO₃ (500 mL). CHCl₃ (300 mL) was added and after 30 min ofvigorous stirring the mixture was transferred to a separatory funnel.The phases were separated and the aq-phase was extracted with CHCl₃(3×250 mL). The combined organic phases were washed with sat. aqNaHCO₃:brine (1:1, 500 mL), dried (Na₂SO₄), filtered and evaporated invacou to give a red foam. The product was purified by Dry Column VacuumChromatography (Ø 10 cm, 50-100% EtOAc in n-heptane v/v, 10% increments,2×100 mL fractions, followed by: 1-10% MeOH in EtOAc v/v, 1% increments,100 ml fractions). The fractions containing nucleoside 75 were pooledand evaporated in vacou to give a white foam (15.6 g, 65%). Further wasisolated the N7 isomere (2.0 g). Compound 75: R_(f)=0.59 (10% MeOH inEtOAc, v/v), ESI-MS m/z found 620.1; 622.0 ([MH]⁺, calcd. 620.1). ¹H NMR(CDCl₃, 400 MHz): δ 8.03 (s, 1H, H8), 7.38-7.29 (m, 5H, Ar—H), 6.14 (d,1H, J=3.3 Hz, H1′), 5.90 (dd (looks like t), 1H, J=3.3 Hz and 3.0 Hz,H2′), 5.29 (s br, 2H, NH₂), 4.78 (d, 1H, J=10.6 Hz, CH₂), 4.70 (d, 1H,J=11.3 Hz, CH₂), 4.67 (d, 1H, J=11.8 Hz, CH₂), 4.44 (d, 1H, J=11.3 Hz,CH₂), 4.37 (d, 1H, J=10.6 Hz, CH₂), 4.37 (d, 1H, J=3.0 Hz, H3′), 3.01(s, 3H, Ms), 2.96 (s, 3H, Ms), 2.14 (s, 3H, Ac). ¹³C NMR (CDCl₃, 100MHz): δ 169.4 (CH₃ C(O)), 159.1, 153.2, 151.7 (C2, C4, C6), 140.4 (C8),136.5, 128.8, 128.5, 128.4 (Ph), 125.3 (C5), 87.0 (C1′), 85.4 (C4′),81.2 (C3′), 78.8 (C2′), 73.4 (CH₂), 67.5, 65.8 (2×CH₂), 37.7, 37.6(2×Ms), 20.6 (CH₃C(O)). Anal. calcd. for C₂₂H₂₆ClN₆O₁₀S₂: C, 42.6; H,4.2; N, 11.3. Found: C, 42.5; H, 4.2; N, 11.0.

9-(3-O-benzyl-5-O-methanesulfonyl-4-C-methanesulfonyloxymethyl-β-L-threo-furanosyl)-2-amino-6-chlorpurine(76)

Compound 75 (5.0 g, 8.1 mmol) is dissolved in methanol (100 mL) andcooled to 0° C. and sat. methanolic ammonia (100 ml) was added. Themixture was stirred at 0° C. for 1 h and then the reaction was quenchedby neutralisation with glacial acetic acid (app. 30 mL). Sat. aq NaHCO₃(100 mL) and CHCl₃ (100 mL) was added and after 5 min of vigorousstirring the mixture was transferred to a separatory funnel. The phaseswere separated and the aq-phase was extracted with CHCl₃ (3×200 mL). Thecombined organic phase was dried (Na₂SO₄), filtered and the solventremoved in vacuo to afford 76 (4.60 g, 99%) as a white solid. R_(f)=0.67(EtOAc). ESI-MS m/z found 578.1; 580.0 ([MH]⁺, calcd. 578.1). ¹H NMR(CD₃CN, 400 MHz): δ 8.03 (s, 1H, H8), 7.41-7.33 (m, 5H, Ar—H), 5.86 (d,1H, J=6.2 Hz, H1′), 5.71 (s br, 2H, NH₂), 5.90 (“q”, 1H, J=4.6 Hz, H2′),4.82 (d, 1H, J=11.5 Hz, CH₂), 4.72 (d, 1H, J=11.5 Hz, CH₂), 4.68 (d, 1H,J=11.0 Hz, CH₂), 4.44-4.32 (m, 5H, CH₂(3), H³′, OH), 3.10 (s, 3H, Ms),2.98 (s, 3H, Ms). ¹³C NMR (CD₃CN, 100 MHz): δ 160.6, 154.7, 151.5, 142.3(C2, C4, C6, C8), 138.4, 129.3, 129.0, 128.9 (Ph), 125.8 (C5), 88.4(C1′), 83.8, 83.6 (C2′, C4′), 77.5 (C3′), 73.9 (CH₂), 69.6, 69.5(2×CH₂), 37.7, 37.5 (2×Ms). Anal. calcd. for C₂₂H₂₆ClN₅O₁₀S₂: C, 42.6;H, 4.2; N, 11.3. Found: C, 42.5; H, 4.2; N, 11.0.

9-(3-O-benzyl-5-O-methanesulfonyl-4-C-methanesulfonyloxymethyl-2-O-trifluoromethanesulfonyl-β-L-threo-furanosyl)-2-amino-6-chlorpurine(77)

Compound 75 (4.50 g, 7.8 mmol) was dissolved in anh. CH₃CN (2×50 mL) andconcentrated in vacuo to remove water traces. The compound was dissolvedin anh. dichloromethane (50 mL) and anh pyridine (6.30 mL, 77.8 mmol)was added followed by the addition of DMAP (3.80 g, 31.1 mmol). Aftercooling to 0° C. trifluoromethanesulfonic anhydride (2.57 mL, 15.6 mmol)was added dropwise during 20 min. The reaction mixture was stirred foran additional 40 min and ice cooled sat. aq NaHCO₃ (100 mL) was addedand after 5 min of vigorous stirring the mixture was transferred to aseparatory funnel. The phases were separated and the aq phase wasextracted with CH₂Cl₂ (2×100 mL). The combined organic phase was washedsuccessively with aq HCl (0.1 M, 2×100 mL) and sat. aq NaHCO₃ (100 mL),dried (Na₂SO₄), filtered and evaporated in vacuo. The residue waspurified by DCVC (Ø=6 cm, 0-100% EtOAc in n-heptane v/v, 5% increments,100 mL fractions) yielding nucleoside 77 (5.05 g, 91%) as a whitepowder. R_(f)=0.18 (1:1 EtOAc in n-heptane v/v). ESI-MS m/z found 710.0;711.9 ([MH]⁺, calcd. 710.0). ¹H NMR (DMSO-d₆, 400 MHz): δ 8.45 (s, 1H,H8), 7.42-7.36 (m, 5H, Ar—H), 7.16 (br. s, 2H NH₂), 6.48-6.48 (m, 2H),5.02 (dd, 1H, J=6.2, 1.6 Hz), 4.85 (dd, 2H, J=10.8, 1.1 Hz), 4.67 (d,1H, J=11.0 Hz), 4.57-4.48 (m, 3H), 3.34 (s, 3H, Ms), 3.18 (s, 3H, Ms).¹³C NMR (DMSO-d₆, 100 MHz): δ 160.0, 153.8, 150.2, 141.2 (C2, C4, C6,C8), 136.4, 128.5, 128.5, 128.4 (Ph), 123.4 (C5), 117.7 (q, J=319.7 Hz,CF₃), 87.0 (C1′), 80.8, 80.2 (C3′, C4′), 73.8 (CH₂), 68.6, 68.4 (2×CH₂),59.8 (C2′), 36.9, 36.5 (2×Ms). Anal. calcd. for C₂₁H₂₃ClF₃N₅O₁₁S₃.0.25EtOAc: C, 36.1; H, 3.4; N, 9.6. Found: C, 36.1; H, 3.2; N, 9.5. NOTE:¹⁹F was also recorded and showed only a single peak.

(1S,3R,4S,7R)-7-benzyloxy-1-(mesyloxymethyl)-3-(guanin-9-yl)-2,5-dioxabicyclo[2.2.1]heptane(78)

3-Hydroxypropionitrile (3.55 mL, 52 mmol) was dissolved in anh. THF (75mL) and cooled to 0° C. Sodiumhydride (60% in mineral oil, 2.50 g, 62.4mmol) was added in portions and the temperature was allowed to raise tort and the mixture was stirred for 30 min at rt. The reaction mixturewas cooled to 0° C. again and9-(3-O-benzyl-5-O-methanesulfonyl-4-C-methanesulfonyloxynnethyl-2-O-trifluoromethanesulfonyl-β-L-threo-furanosyl)-2-amino-6-chlorpurine(77) (7.37 g, 10.4 mmol) dissolved in anh. THF (75 mL) was addeddropwise over 20 min and the temperature was allowed to raise to rt.After 8 h the reaction was quenched by addition of HCl (1 M, aq):brine(1:9, 250 mL) and the mixture was transferred to a separatory funnel.The phases were separated and the aq.-phase was extracted with EtOAc(3×200 mL). The combined organic phases were dried (Na₂SO₄), filteredand evaporated in vacuo to give a red oil. The product was purified byfirst filtration through a short silica plug (Ø 6 cm, 10% MeOH in EtOAcv/v, 500 mL) and the resulting material was then precipitated fromEtOH:H₂O (1:1) resulting in 78 as a tan solid (4.64 g, 96%). R_(f)=0.31(10% MeOH in EtOAc v/v); ESI-MS m/z found 464.1 ([MH]⁺, calcd. 464.1);¹H NMR (DMSO-d₆, 400 MHz): δ=10.63 (s br, 1H, NH), 7.72 (s, 1H, H8),7.30-7.24 (m, 3H, Ar—H), 7.16-7.11 (m, 2H, Ar—H), 6.65 (s br, 2H, NH₂),5.86 (s, 1H, H1′), 4.83 (d, 1H, J=11.5 Hz, H1″), 4.71 (d, 1 H, J=11.4Hz, H1″), 4.60 (d, 1H, J=1.8 Hz, H2′/H3′), 4.52 (d, 1H, 3=11.9 Hz, PhCH₂), 4.34 (d, 1H, J=11.9 Hz, PhCH ₂), 4.27 (d, 1H, J=1.8 Hz, H2′/H3′),4.08 (d, 1 H, J=8.4 Hz, H5′), 3.86 (d, 1H, J=8.2 Hz, H5′), 3.27 (s, 3H,Ms); ¹³C NMR (DMSO-d₆, 100 MHz): δ=156.7 (C6), 153.8 (C2), 150.5 (C4),137.0 (Ph), 135.6 (C8), 128.3, 127.9, 127.9 (Ph), 116.2 (C5), 86.8(C4′), 85.5 (C1′), 80.2 (C3′), 76.4 (C2′), 72.5, 72.2 (Ph CH₂, C5′),66.4 (C1″), 36.8 (Ms). Anal. calcd. for C₁₉H₂₁N₅O₂S: C, 49.2; H, 4.6; N,15.1. Found: C, 49.4; H, 4.5; N, 15.2.

(1S,3R,4S,7R)-7-benzyloxy-1-(benzoyloxymethyl)-3-(guanin-9-yl)-2,5-dioxabicyclo[2.2.1]heptane(79)

Compound 78 was dissolved in DMSO (25 mL) and BzONa (2.22 g, 15.24 mmol)was added. Heated to 100° C. for 6 h and then to 120° C. for 3 h. Thereaction was diluted with EtOAc (50 mL) and quenched with water:sat. aq.NaHCO₃ (1:1, 100 mL). The phases were separated and the aq phase wasextracted with EtOAc (3×50 mL). The comb. org. phases were washed withBrine (2×100 mL) dried (Na₂SO₄), filtered and concentrated. The productwas purified by Dry Column Vacuum Chromatography (Ø 4 cm, 0-15% MeOH indichloromethane v/v, 1% increments, 100 mL fractions). The fractionscontaining nucleoside 79 were pooled and evaporated in vacuo to give awhite solid (1190 mg, 95%). R_(f)=0.15 (5% MeOH in DCM v/v); ESI-MS m/zfound 488.3 ([M−H]⁻, calcd. 488.2); ¹H NMR (DMSO-d₆, 400 MHz): δ=10.63(s, 1H, NH), 7.95 (d, 2H, J=7.3 Hz, Bz), 7.67 (s, 1H, H8), 7.64 (t, 1H,J=7.3 Hz, Bz), 7.50 (t, 3H, J=7.3 Hz, Bz), 7.24-7.18 (m, 3H, Bn),7.12-7.06 (m, 2H, Bn), 6.54 (br s, 2H. —NH₂), 5.86 (s, 1H, H1′), 4.79(s, 2H, H1″), 4.59 (d, 1H, J=1.8 Hz, H2′/H3′), 4.49 (d, 1H, J=11.9 Hz,PhCH ₂O), 4.34 (d, 1H, J=11.9 Hz, PhCH ₂O), 4.29 (d, 1H, J=1.8 Hz,H2′/H3′), 4.11 (d, 1H, J=8.4 Hz, H5′ a), 3.93 (d, 1H, J=8.2 Hz, H5′ b);¹³C NMR (DMSO-d₆, 100 MHz): δ=165.3 (C(O)Ph), 156.7, 153.8, 150.1 (C2,C4, C6), 137.0 (Bn), 135.5 (C8), 133.7 (Bz), 129.4, 129.1, 128.9, 128.3,127.9 (Ph), 116.3 (C5), 86.8, 85.9 (C4′, C1′), 80.0, 76.4 (C2′, C3′),72.7, 72.2 (Ph CH ₂, C5′), 60.6 (C1″)

(1R,3R,4S,7R)-3-(guanin-9-yl)-7-hydroxy-1-hydroxymethyl-2,5-dioxabicyclo-[2.2.1]heptane(80)

Compound 79 (2.33 g, 4.16 mmol) was suspended in MeOH (100 mL) andPd(OH)₂—C (20%, 292 mg, 10% mol Pd) and ammonium formiat (5.24 g; 83.2mmol) were added. The mixture was heated to reflux. After 4 h furtherPd(OH)₂—C (20%, 292 mg, 10% mol Pd) was added and after an additional 4h the last Pd(OH)₂—C (20%, 292 mg, 10% mol Pd) was added. Reflux foranother 12 h.

The catalysis was removed by filtration through paper, the filter paperwith catalyst was boiled in MeOH for 30 min and then filter again. Thetwo methanolic solutions are pooled and filtered through Celite. TheCelite was washed thoroughly with hot MeOH. All the methanolic solutionswere pooled and concentrated. Taken up in H₂O and lyophilized twiceresulted in 79 as white solid. (1100 mg, 90%). R_(f)=0.01 (10% MeOH inEtOAc v/v); ESI-MS m/z found 296.1 ([MH]⁺, calcd. 296.1); ¹H NMR (D₂O,400 MHz): δ=7.90 (s, 1H, H8), 5.91 (s, 1H, H1′), 4.74 (d, 1H, J=2.4 Hz,H2′/H3′), 4.40 (d, 1H, J=2.4 Hz, H2′/H3′), 4.12 (d, 1H, J=8.6 Hz, H5′),4.02 (d, 1H, J=8.7 Hz, H5′), 4.01 (s, 2H, H5′); ¹³C NMR (D₂O, 100 MHz):δ=158.7 (C6), 153.7 (C2), 150.8 (C4), 138.4 (C8), 115.4 (C5), 88.7(C4′), 86.3 (C1′), 78.1 (C3′), 73.2, 72.4 (C2′, C5′), 57.4 (C1″); Anal.calcd. for C₁₁H₁₃N₅O₅.H₂O: C, 42.2; H, 4.8; N, 22.4. Found: C, 42.0; H,4.5; N, 22.2.

(1S,3R,4S,7R)-1-(4,4′-dimethoxytrityloxymethyl)-3-(2-N-((dimethylamino)methylidene)-7-hydroxy-guanin-9-yl)-2,5-dioxabicyclo[2.2.1]heptane(82)

Compound 80 (860 mg, 2.91 mmol) was dissolved in anh. DMF (25 mL) andN,N-Dimethylformamide dimethyl acetal (0.77 mL, 5.83 mmol) was added.After 4 h the reaction was completed and most of the DMF was remove invacuo. The resulting slurry 81 was coevaporated twice from anh. pyridine(2×25 mL) and suspended in anh. pyridine (10 mL).4,4′-dimethoxytritylchloride (1.48 g, 4.37 mmol) was added and thereaction mixture was stirred for 16 h at rt. Most of the pyridine wasremoved in vacuo and the residue was taken up in DCM (50 mL) and washedwith half sat. aq. NaHCO₃ (2×50 mL) and brine (50 mL). The comb. aq.phases were extracted with DCM (2×50 mL) and the comb. org. phases weredried (Na₂SO₄), filtered and evaporated in vacuo to give a yellow foam.The product was purified by Dry Column Vacuum Chromatography (Ø 4 cm,0-10% MeOH in DCM v/v, 0.5% increments, 100 ml fractions). The fractionscontaining nucleoside 82 were pooled and evaporated in vacuo to give awhite foam (1.10 g, 58%). R_(f)=0.08 (10% MeOH in EtOAc, v/v); ESI-MSm/z found 653.3 ([MH]⁺, calcd. 653.3; ¹H NMR (DMSO, 400 MHz): δ=11.29(s, 1H, NH), 8.57 (s, 1H, N═CH), 7.87 (s, 1H, H8), 7.46-7.40 (m, 2H,DMT), 7.35-7.22 (m, 7H, DMT), 6.93-6.88 (m, 4H, DMT), 6.00 (s, 1H, H1′),5.92 (d, 1H, J=3.8 Hz, H2′), 4.51 (d, 1H, J=2.0 Hz, OH), 4.21 (dd, 1H,J=3.5, 2.2 Hz, H3′), 4.05 (d, 1H, J=8.2 Hz, H1″), 3.98 (d, 1H, J=8.2 Hz,H1″), 3.74 (s, 6H, OCH₃), 3.51 (d, 1H, J=10.2 Hz, H5′), 3.38 (d, 1H,J=10.2 Hz, H5′), 3.33 (s, 3H, NCH₃), 3.15 (s, 3H, NCH₃); ¹³C NMR (DMSO,100 MHz): δ 158.0 (DMT), 157.8, 157.4, 157.1 (C2, C6, N═CH), 149.2 (C4),144.5 (DMT), 137.3 (C8), 135.2 (DMT), 129.6, 129.6, 127.7, 127.5, 126.6(DMT), 118.9 (C5), 113.1 (DMT), 87.3, (C4′), 86.1 (C1′), 85.5 (DMT),78.1 (C3′), 73.0, 72.7 (C1″, C2′), 60.0 (C5′), 54.9 (OCH₃), 40.5, 34.6(N(CH₃)₂); Anal. calcd. for C₃₅H₃₆N₆O₇.H₂O: C, 62.7; H, 5.7; N, 12.5.Found: C, 62.8; H, 5.4; N, 12.6.

(1S,3R,4S,7R)-1-(4,4′-dimethoxytrityloxymethyl)-7-(2-cyanoethoxy(diisopropylamino)phosphinoxy)-3-[2-N—((N′,N′-dimethylamino)methylidene)-guanin-9-yl]-2,5-dioxabicyclo[2.2.1]heptane(83)

Compound 82 (750 mg, 1.15 mmol) was dissolved in anh DMF (25 mL) and4,5-dicyanoimidazole in MeCN (1.0 M, 0.80 mL, 0.8 mmol) was added atambient temperature with stirring.2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.40 mL, 1.26mmol) was added dropwise to the reaction mixture. After 4 h the reactionwas diluted with EtOAc (70 mL) and transferred to a separatory funneland extracted with sat. aq NaHCO₃ (2×50 mL) and brine (50 mL). Thecombined aq phases were extracted with EtOAC (100 mL). The organicphases were pooled and dried (Na₂SO₄). After filtration the organicphase was evaporated in vacuo to give a yellow foam. Purification byDCVC (Ø 2 cm, 1-10% MeOH, EtOAc, v/v, 0.5% increments, 50 mL fractions(the column was pretreated with 1% Et₃N in EtOAc v/v)) afforded amidite83 (480 mg, 49%) as a white solid. R_(f)=0.50 (1%, TEA, 10% MeOH in DCMv/v/v); ESI-MS m/z found 853.2 ([MH]⁺, calcd. 853.4); ³¹P NMR (CDCl₃ 121MHz) δ 151.7, 150.3.

(1S,3R,4S,7R)-7-benzyloxy-3-(2-amino-6-chloro-purine-9-yl)-1-(methanesulfonyloxymethyl)-2,5-dioxabicyclo[2.2.1]heptane(84)

Compound 77 (7.44 g, 10.4 mmol) was dissolved in THF (300 mL). Aftercooling to 0° C. aq LiOH (1.0M, 105 mL) was added. The reaction mixturewas stirred at 0° C. for 4 h and then for additional 2 h at rt. Thereaction was quenched by addition of aq HCl (1.0M, sat. with NaCl, 100mL) and after 5 min of vigorous stirring the mixture was transferred toa separatory funnel. The phases were separated and the aq-phase wasextracted with EtOAc (3×150 mL). The combined organic phase was washedwith brine:sat. aq NaHCO₃ (1:1, 200 mL), dried (Na₂SO₄), filtered andevaporated in vacuo. The residue was purified by DCVC (Ø=6 cm, 50-100%EtOAc in n-heptane v/v, 5% increments, 2×100 mL fractions) yieldingnucleoside 84 (4.49 g mg, 89%) as a white powder. R_(f)=0.49 (20%n-heptane in EtOAc (v/v)). ESI-MS m/z found 482.1.; 484.0 ([MH]⁺, calcd.482.1). ¹H NMR (DMSO-d₆, 400 MHz): δ 8.09 (s, 1H, H8), 7.26-7.19 (m, 3H,Ar—H), 7.08-7.04 (m, 2H, Ar—H), 7.01 (br. s, 2H, NH₂), 5.96 (s, 1H,H1′), 4.86 (d, 1H, J=11.3 Hz, H5″), 4.76 (d, 1H, J=11.3 Hz, H5″), 4.65(d, 1H, J=2.0 Hz, H2′), 4.51 (d, 1H, J=11.9 Hz, CH₂), 4.32 (d, 1H,J=11.7 Hz, CH₂), 4.31 (d, 1H, J=2.0 Hz, H3′), 4.10 (d, 1H, J=8.2 Hz,CH₂), 3.89 (d, 1H, J=8.4 Hz, CH₂), 3.28 (s, 3H, Ms). ¹³C NMR (DMSO-d₆,100 MHz): δ 159.6, 153.0, 149.0 (C2, C4, C6), 140.8 (C8), 136.7, 128.0,127.8, 127.7 (Ph), 123.2 (C5), 86.8 (C4′), 85.6 (C1′), 80.0 (C3′), 75.8(C2′), 72.3, 72.2 (C5′, CH₂Ph), 66.2 (C5″), 36.6 (Ms).

(1S,3R,4S,7R)-7-benzyloxy-3-(2-amino-6-chloro-purine-9-yl)-1-(benzoyloxymethyl)-2,5-dioxabicyclo[2.2.1]heptane(85)

Compound 84 (4.49 g, 9.32 mmol) was dissolved in DMSO (200 mL) and BzONa(6.76 g, 46.6 mmol) was added. The reaction was stirred at 100° C. for16 h. The reaction allowed to cool to room temperature and EtOAc (200mL) and brine: sat. aq NaHCO₃ (1:1, 400 mL) was added. The mixture wastransferred to a separatory funnel. The phases were separated and theaq-phase was extracted with EtOAc (3×200 mL). The combined organic phasewas washed with brine (half-sat., 2×200 mL), dried (Na₂SO₄), filteredand evaporated in vacuo. The residue was purified by DCVC (Ø=6 cm,50-100% EtOAc in n-heptane v/v, 5% increments, 2×100 mL fractions, 0-10%MeOH in EtOAc v/v, 1% increments, 100 mL) yielding nucleoside 85 (3.30g, 70%) as a white powder. R_(f)=0.40 (35% n-heptane in EtOAc (v/v)).ESI-MS m/z found 508.2.; 510.1 ([MH]⁺, calcd. 508.1). ¹H NMR (DMSO-d₆,400 MHz): δ 8.05 (s, 1H, H8), 7.98 (d, 2H, J=7.3 Hz, Bz), 7.68 (t, 1H,J=7.3 Hz, Bz), 7.53 (t, 2H, J=7.7 Hz, Bz), 7.25-7.15 (m, 3H, Bn),7.05-7.00 (m, 4H, Bn, NH₂), 5.98 (s, 1H, H1′), 4.85 (s, 2H, H5″), 4.67(d, 1H, J=1.8 Hz, H2′), 4.50 (d, 1H, J=12.1 Hz, CH₂), 4.35 (d, 1H, J=2.0Hz, H3′), 4.34 (d, 1H, J=11.7 Hz, CH₂), 4.16 (d, 1H, J=8.4 Hz, CH₂),3.98 (d, 1H, J=8.1 Hz, CH₂). ¹³C NMR (DMSO-d₆, 100 MHz): δ 165.1 (PhCO),159.6, 152.9, 149.0 (C2, C4, C6), 140.6 (C8), 136.7, 133.5, 129.2,128.9, 128.7, 128.0, 127.9, 127.7, 127.6 (Ph), 123.2 (C5), 86.7 (C4′),85.9 (C1′), 79.9 (C3′), 75.8 (C2′), 72.5, 72.1 (5′, CH₂Ph), 60.4 (C5″).

9-(3-O-Benzyl-2-deoxy-2-iodo-5-O-methanesulfonyl-4-C-(methanesulfonyloxymethyl)-β-D-threo-pentofuranosyl)-6-N-benzoyladenine(87)

9-(3-O-Benzyl-5-O-methanesulfonyl-4-C-(methanesulfonyloxymethyl)-2-O-trifluoromethanesulfonyl-β-D-erythro-pentofuranosyl)-6-N-benzoyladenine(589 mg, 0.755 mmol) was dissolved in dry acetonitrile (15 ml), addedlithiumiodide (202 mg, 2 equiv.) and heated to reflux. After 2 hrs, LCMSshows full conversion. Solvent was removed in vacuo and the residue waspartitioned between DCM (50 ml) and water (50 ml). Layers were separatedand the organic layer was washed with brine (50 ml), dried (Na₂SO₄),filtered and the solvent removed in vacuo to afford an orange foam (514mg, 90% yield)

9-(2-azido-3-O-Benzyl-2-deoxy-5-O-methanesulfonyl-4-C-(methanesulfonyloxymethyl)-β-D-erythro-pentofuranosyl)-6-N-benzoyladenine(88)

Compound 87 (30 mg) was dissolved in DMF/water 1:1 (2 ml) and followedby sodium azide (26 mg, 10 equiv.). The reaction mixture stirred at 80°C. overnight. LCMS confirms conversion of starting material to productsubstituted with azide.

The invention claimed is:
 1. A method for the synthesis of a compound of the general formula IV

wherein X is —O— Z is —CH₂ B is a nucleobase; R³ is selected from —R^(H), —N₃, —NR^(H)R^(H*) , —NR^(H)C(O)R^(H*,) —C(O)NR^(H)R^(H*,) —OR^(H), —OC(O)R^(H), —C(O)OR^(H), —SR^(H), —SC(O)R^(H), and tri(C₁₋₆-alkyl/aryl)silyloxy; each R^(H) and R^(H*) independently being selected from hydrogen, optionally substituted C₁₋₆-alkyl, optionally substituted aryl, and optionally substituted aryl-C_(i-6)-alkyl; A⁴ and A⁵ independently are selected from C₁₋₆-alkylene; and R⁵ is selected from iodo, bromo, chloro, C₁₋₆-alkylsulfonyloxy optionally substituted with one or more substituents selected from halogen and phenyl optionally substituted with one or more substituents selected from nitro, halogen and C₁₋₆-alkyl, and arylsulfonyloxy optionally substituted with one or more substituents selected from nitro, halogen, C₁₋₆-alkyl, and C₁₋₆-alkyl substituted with one or more halogen; said method comprising the following steps: treating an intermediate of the general formula I:

wherein X, B, R³, A⁴, and A⁵ are as defined above; R² is selected from iodo, C₁₋₆-alkylsulfonyloxy optionally substituted with one or more substituents selected from halogen and phenyl optionally substituted with one or more substituents selected from nitro, halogen and C₁₋₆-alkyl, and arylsulfonyloxy optionally substituted with one or more substituents selected from nitro, halogen, C₁₋₆-alkyl, and C₁₋₆-alkyl substituted with one or more halogen; R³ and R² may together form an epoxide and R⁴ and R⁵ independently are as defined for R⁵ above, or R⁴ and R⁵ together constitutes a tetra(C₁₋₆-alkyl)disiloxanylidene group; with a nucleophile selected from organometallic hydrocarbyl radicals, so as to substitute R², and effecting ring-closure between the C2′ and C4′ positions so as to yield the LNA analogue of the formula IV.
 2. The method according to claim 1, wherein R² is selected from C₁₋₆-alkylsulfonyloxy optionally substituted with one ore more halogen, and arylsulfonyloxy optionally substituted with one or more substituents selected from nitro, halogen, C₁₋₆-alkyl, and C₁₋₆-alkyl substituted with one or more halogen; R³ is optionally substituted aryl(C₁₋₆-alkyl)oxy; and R⁴ and R⁵ are independently selected from C₁₋₆-alkylsulfonyloxy optionally substituted with one or more halogen an arylsulfonyloxy optionally substituted with one or more substituents selected from nitro, halogen, C₁₋₆-alkyl, and C₁₋₆-alkyl substituted with one or more halogen.
 3. The method according to claim 1, wherein A⁴ and A⁵ are methylene.
 4. The method according to claim 1, wherein R⁴ and R⁵ are identical.
 5. The method according to claim 1, wherein B is selected from adenine, guanine, 2,6-diaminopurine, thymine, 2-thiothymine, cytosine, methyl cytosine, uracil, 5-fluorocytosine, xanthine, 6-aminopurine, 2-aminopurine, 6-chloro 2-amino-purine, and 6-choropurine, R² is selected from C₁₋₆-alkylsulfonyloxy substituted with one or more halogen, R³ is benzyl, and R⁴ and R⁵ are independently selected from C₁₋₆-alkylsulfonyloxy optionally substituted with one or more substituents selected from halogen and C₁₋₆-alkyl substituted with one or more halogen.
 6. The method according to claim 1, wherein R⁴ and R⁵ are independently selected from methanesulfonyloxy, trifluoromethanesulfonyloxy, ethanesulfonyloxy, 2,2,2,-trifluoroethanesulfonyloxy, propanesulfonyloxy, iso-propanesulfonyloxy, butanesulfonyloxy, nonafluorobutanesesulfonyloxy, pentanesulfonyloxy, cyclopentanesulfonyloxy, hexanesulfonyloxy, cyclohexanesulfonyloxy, 2-chloro-α-toluenesulfonyloxy, ortho-toluensulfonyloxy, meta-toluenesulfonyloxy, para-toluenesulfonyloxy, benzenesulfonyloxy, ortho-bromobenzenesulfonyloxy, meta-bromobenzenesulfonyloxy, para-bromobenzenesulfonyloxy, ortho-nitrobenzenesulfonyloxy, meta-nitrobenzenesulfonyloxy, and para-nitro-benzenesulfonyloxy.
 7. The method according to claim 1, wherein the intermediate has the formula III

wherein B, R⁴ and R⁵ are as defined in claim 1, and wherein R³ is —OR^(H) or —OC(O)R^(H), where R^(H) is as defined in claim
 1. 8. The method according to claim 1, wherein B is selected from adenine, guanine, 2,6-diaminopurine, thymine, 2-thiothymine, cytosine, methyl cytosine, uracil, 5-fluorocytosine, xanthine, 6-aminopurine, 2-aminopurine, 6-chloro-2-amino-purine, and 6-chloropurine, R³ is benzyl, and R⁴ and R⁵ are both methylsulfonyloxy. 